Antibodies directed against signal peptides, methods and uses thereof

ABSTRACT

The invention concerns methods employing antibodies directed against the signal peptide (SP) domain of various disease-associated polypeptides. These anti SP antibodies are capable of detecting cell surface expression of these SP domains and therefore they can be used in methods of diagnosis and/or therapy. In one aspect, the invention provides a method for determining the suitability for treatment of a subject suffering from a disease, whereby detection of cell surface expression of a specific SP indicates that the subject would benefit from therapy directed against this SP. The invention is specifically exemplified with antibodies directed against the signal peptide of MUC1 which is expressed on the surface of various cancer cells, and with signal peptide domains of  Mycobacterium tuberculosis.    
     In other aspects the invention provides methods for diagnosis of disease based on the detection of endogenously produced anti SP antibodies.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/344,837 filed on Mar. 13, 2014, which is a National Phase of PCTPatent Application No. PCT/IL12/050365 having International filing dateof Sep. 13, 2012, which claims the benefit of priority of U.S. PatentApplication No. 61/535,017 filed on Sep. 15, 2011. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

The Sequence Listing submitted m text format (.txt), named“SequenceListing.txt”, created on Aug. 14, 2017, 4.87 KB, isincorporated herein by reference.

TECHNOLOGICAL FIELD

This invention relates to diagnostic and therapeutic methods employingantibodies directed against the signal peptide (SP) of disease-relatedpolypeptides, in particular for the diagnosis and treatment of cancerand infectious diseases, specifically Tuberculosis.

BACKGROUND

In both prokaryotic and eukaryotic cells, proteins destined (ortargeted) for secretion or for insertion into cellular membranes useshort—13-50 amino acids long, usually amino-terminal, signal peptides(SP), also termed signal sequences.

Different SPs of various antigens exhibit high sequence variability withno particular sequence identity while conforming to the motif needed tomaintain their functionality (Lyko F. et al., J Biol Chem 1995,270:19873-19878). Recently, SP and trans-membrane domains were found tohave exceptionally high major histocompatibility complex (MHC) class Iand II epitope densities. The improved MHC binding of these domainsrelies on their hydrophobic nature and, in SP, also on their specificsequence (Kovjazin, R. et al., Mol Immunol 2011, 48: 1009-1018).

The post targeting function of SP (Martoglio B. et al., Biochem SocTrans 2003, 31:1243-1247), suggests that SP fragments may be directed tothe cell membrane or to the extracellular compartment even withoutassociation with MHC molecules.

WO 2008/035350 relates to SP-derived vaccines, capable of inducing arobust, antigen specific T-cell immunity and which are applicable to themajority of the population.

Tumor associated antigens (TAA) are cancer associated epitopes or markerproteins. Many such antigens are known in the art, for example MUC1.

MUCl, the polymorphic epithelial mucin, is a glycoprotein with fewalternative splicing variants encoding for transmembranal andsecreted/soluble products, both are expressed in abroad range of tumors(Graham, R. A et al., Cancer Immunol Immunother 1996, 42:71-80 and Ho,S. B. et al., Cancer Res 1993, 53:641-651). MUCl is one of the few knowntargets that are expressed in more than 90% of the common solid tumorcancers, which include, among others: Colon, Gastric, Lung, Renal Cell(RC), Transitional Cell (TC), Prostate, Pancreas, Breast, Ovary andThyroid. It is also associated with many non-solid tumors among which:Lymphoma, Leukaemia and Multiple Myeloma (MM).

It has been shown that the copy number of MUC1 increases in primaryinvasive breast carcinomas compared with normal tissue in correlationwith its protein expression (Lacunza, E. et al., Cancer Genet Cytogenet2010, 201:102-110). Other studies, mainly in MM, have demonstrated thatMUC1 is expressed on the cell surface of most MM cell lines, bone marrow(BM) plasma cells derived from patients, and plasmacytomas (Treon, S. P.et al., Blood 2000, 96:3147-3153).

Soluble MUCl (sMUCl) levels containing an extracellular tandem repeatarray (TRA) were reported to correlate with tumor mass, as measured byFDA (US Food and Drug Administration) approved assays like CA15.3 inbreast cancer, and/or CA27.29 in MM cancer (Croce, M. V. et al., BreastCancer Res Treat 2003, 81:195-207 and Teron, S. P et al., Blood, 2000,96: 3147-53). However, despite the wide tumor distribution of MUCl, useof the CA15.3 and CA27.29 markers is confined to monitoring theprognosis and response to treatment in patients with advanced breastcancer (Gion, M. et al Clin. Chem. (1999) 45:630-637) and it is notsensitive enough to be used for early diagnosis. In contrast, naturallygenerated autoantibodies to TAAs are detectable even before the tumor isclinically apparent (Lu H. et al J. Proteome Res. (2008) 7: 1388-1394).

The use of anti-MUC1 monoclonal antibodies, mostly directed against theextracellular TRA epitope, was also reported as an anti-cancer modality(Tang, C. K. et al., Expert Rev Vaccines 2008, 7:963-975). However,since these epitopes are not restricted to cell surface expression butare also expressed in the sera of patients, the use of these anti-MUC1monoclonal antibodies for diagnosis and therapy may be of reducedpotency.

EP 1137943 relates to an in vitro method for detecting the presence of acancer associated marker protein in mammals, where the cancer associatedmarker may be, among others, a modified cancer-associated form of MUCl.

GENERAL DESCRIPTION

The present invention is based on the finding that antibodies that weregenerated against the signal peptide (SP) domain of disease-associatedpolypeptides were capable of detecting cell surface expression of theseSP domains in cancer cells or bacterial cells.

Therefore, in a first of its aspects, the present invention provides amethod for determining the suitability for treatment of a subjectsuffering from a disease, the method comprises:

-   -   a. contacting a biological sample containing cells obtained from        said subject with an antibody directed against a signal peptide        (SP), or any fragment thereof, of a polypeptide associated with        said disease; and    -   b. determining the expression level of said SP, or any fragment        thereof, on the surface of cells in said biological sample,        wherein the presence of said SP, or any fragment thereof, on the        surface of said cells in a level higher than a control level        indicates that said subject is suitable for treatment.

In another aspect, the present invention provides a method of treatmentof a subject suffering from a disease, the method comprisesadministering to said subject a therapeutically effective amount of atleast one antibody directed against a signal peptide (SP), or anyfragment thereof, of a polypeptide associated with said disease.

In yet another aspect, the present invention provides a method ofdiagnosing a disease in a subject, the method comprises:

-   -   a. contacting a biological sample containing cells obtained from        said subject with an antibody directed against a signal peptide        (SP), or any fragment thereof, of a polypeptide associated with        said disease; and    -   b. determining the level of said SP, or any fragment thereof, on        the surface of cells in said biological sample,        wherein the presence of a level of said SP in said sample which        is higher than a control level is indicative of a disease.

In yet another aspect, the present invention provides an isolatedantibody directed against a signal peptide (SP), or any fragmentthereof, of a polypeptide associated with a disease, for use in a methodof determining the suitability for treatment of a subject suffering fromsaid disease, wherein said method comprises:

-   -   a. contacting a biological sample containing cells obtained from        said subject with said isolated antibody; and    -   b. determining the expression level of said SP, or any fragment        thereof, on the surface of cells in said biological sample,        wherein the presence of said SP, or any fragment thereof, on the        surface of said cells in a level higher than a predetermined        baseline indicates that said subject is suitable for treatment.

In yet another aspect, the present invention provides an isolatedantibody directed against a signal peptide (SP), or any fragmentthereof, of a polypeptide associated with a disease, for use in a methodof treatment of a subject suffering from said disease, wherein saidmethod comprises administering a therapeutically effective amount ofsaid antibody to the subject.

In yet another aspect, the present invention provides an isolatedantibody directed against a signal peptide (SP), or any fragmentthereof, of a polypeptide associated with a disease for use in a methodof diagnosing a disease in a subject, said method comprises:

-   -   a. contacting a biological sample containing cells obtained from        said subject with said isolated antibody; and    -   b. determining the level of said SP, or any fragment thereof, on        the surface of cells in said biological sample,        wherein the presence of a level of said SP in said sample which        is higher than a predetermined baseline level is indicative of a        disease.

In yet another aspect, the present invention provides a method fordetecting a disease in a subject, said method comprises:

-   -   a. contacting a biological sample obtained from said subject        with at least one SP, or any fragment thereof, of polypeptide        associated with said disease; and    -   b. measuring the level of endogenous antibodies directed against        said SP, or any fragment thereof, in said biological sample,        wherein the presence of said endogenous antibodies in the sample        in a level higher than a control is indicative of disease.

In one embodiment, said disease is cancer.

In another embodiment, said disease is a bacterial disease, a fungaldisease, a parasite disease, a prion disease, or a viral disease.

In one embodiment, the polypeptide associated with said disease is aTumor associated antigen (TAA).

In specific embodiments, said TAA is selected from a group consisting ofMUCl, Armet, HSP60, CANX, MTHFD2, FAP, MMP6, BAGE-1, GNTV, Q5H943, CEA,Pmel, Kallikrein-4, Mammaglobin-1, MART-I, GPR143-OA1, PSA, TRPI,Tyrosinase, FGP-5, NEU proto-oncogene, Aft, MMP-2, PSMA,Telomerase-associated protein-2, PAP, Uroplakin II and Proteinase 3.

In one specific embodiment, said TAA is MUC1.

In yet another specific embodiment, the polypeptide associated with saiddisease is MUC1 and said SP, or any fragment thereof, is a peptideselected from a group consisting of the peptides denoted by SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

In certain embodiments, said cancer 1s selected from a group consistingof multiple myeloma (MM), breast cancer, ovary cancer, colon cancer,rectal cancer, gastric cancer, non-small lung carcinoma (NScLC), renalcell carcinoma (RCC), transitional cell carcinoma (TCC), prostatecancer, pancreatic cancer, thyroid cancer, Squamous cell carcinoma(SCC), Thymic Carcinoma, lymphoma, leukemia, and Mesothelioma.

In a specific embodiment, said cancer is multiple myeloma (MM), breastcancer or ovarian cancer.

In yet another embodiment, the disease is a bacterial disease caused byMycobacterium tuberculosis (MTb).

In specific embodiments, the polypeptide associated with the bacterialdisease caused by Mycobacterium tuberculosis (MTb) is selected from thegroup consisting of Antigen 85B, Lipoprotein IpqH, ATP dependenthelicase putative, Uncharacterized protein Rv0476/MTO4941 precursor andUncharacterized protein Rv1334/MT1376 precursor.

In certain embodiments the antibody used in the methods of the inventionis selected from a group consisting of a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a humanized antibody, a humanantibody, or any fragment thereof, which retains the binding activity ofthe antibody.

In specific embodiments said antibody is the polyclonal antibody R23 orits immunoglobulin enriched fraction R23IgG (also termed SPimAb-3A), themonoclonal antibody SPmAb-2.1 or the monoclonal antibody SPmAb-6deposited at the ECACC on Sep. 13, 2012 under Accession no. 12091301.

In one embodiment, the present invention provides a method fordetermining the suitability for treatment of a subject suffering from adisease, as described above, wherein said treatment comprises at leastone of a vaccine capable of eliciting an immune response against saidSP, or any fragment thereof and an agent capable of binding said SP, orany fragment thereof.

In one embodiment, said agent is at least one of an antibody, a ligand,or any fragment thereof which is capable of binding the SP expressed onsaid cells.

In one specific embodiment, the polypeptide associated with said diseaseis MUC1 and said vaccine capable of eliciting an immune response is atherapeutic vaccine comprising MUCl SP.

In certain embodiments the methods of determining the suitability fortreatment of a subject suffering from a disease or the methods ofdiagnosis of the invention described above are performed ex vivo.

In certain embodiments the biological sample used in these methods isselected from a group consisting of plasma, serum, whole blood, urine,sweat, lymph, faeces, cerebrospinal fluid, bone marrow biopsy oraspirate, or nipple aspiration.

In one embodiment the method of treatment of a subject suffering from adisease according to the invention as described above the antibody is aneutralizing antibody.

In yet another embodiment, the antibody is associated with or combinedwith a cytotoxic moiety.

In specific embodiments, said cytotoxic moiety is selected from a groupconsisting of a radioactive agent, a toxin, an anti-metabolite, or analkylating agent.

In yet another aspect, the present invention provides a kit comprising:

-   -   (a) an isolated antibody directed against a signal peptide (SP),        or any fragment thereof, of a polypeptide associated with a        disease;    -   (b) means for detecting the binding of said isolated antibody on        the surface of cells in a biological sample obtained from a        subject; and optionally further comprising    -   (c) instructions for use of said kit.

In certain embodiments said kit further comprises a vaccine comprisingthe signal peptide, or a fragment thereof capable of eliciting an immuneresponse, of said polypeptide associated with a disease.

In certain embodiments, said means comprises

-   -   (a) a detectably-labeled secondary antibody which recognizes the        anti SP antibody; and    -   (b) optionally further comprising reagents for performing said        selection.

In one specific embodiment, said isolated antibody is directed againstMUC1 SP.

In another specific embodiment, said isolated antibody is directedagainst the SP of a polypeptide of Mycobacterium tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIGS. 1AI-1E show characteristics of antibodies that were generatedagainst the SP domain of MUC1. FIGS. 1AI-1AIII show polyclonal antibodytiters in sera obtained from two immunized rabbits, R23 and R32. Variousserum dilutions are presented. Anti MUC1-SP-M is shown in FIG. 1AI;anti-BAGE-SP-L in FIG. 1AII; and anti TB-Rv0476/4941-SP-L in FIG. 1AIII.FIG. 1B is a graph showing the titer of R23 antibodies directed againstvarious MUC1 epitopes. FIG. 1C is a graph showing % inhibition ofbinding of R23 and R32 polyclonal antibodies by various MUC1 and BAGE SPpeptides. FIG. 1D is a graph showing the titer of antibodies in mousehyperimmune sera directed against various MUC1 epitopes. FIG. 1E is agraph showing % inhibition of binding of SPmAb-2. I and SPmAb-6monoclonal antibodies by various MUC1 SP peptides.

FIG. 2A shows photographs of immunofluorescence microscopy staining ofvarious cancer cell types both solid and non-solid (ES-2, OVACAR-3, U266and RPMI 8226) different MUC1 SP antibodies: the control MUC1 TRAmonoclonal antibody H23, monoclonal antibodies (mAbs) SPmAb-2. I andSPmAb-6 and the IgG fraction of the hyper immune polyclonal antibodiesR23. FIG. 2B shows photographs of immunofluorescence microscopy stainingof ES-2 MUC1 negative ovarian cells (untransfected or transfected with aMUC-TM construct containing both alfa and beta sub-units) with theanti-MUC1 SP monoclonal antibody (mAb) SPmAb-2.I and the MUC1 TRA mAbH23.

FIGS. 3A-3B show photographs of specific staining of tumor tissueobtained from breast cancer patient with H23 (FIG. 3A) and SPmAb-2. I(FIG. 3B).

FIGS. 4A-4E show graphical representations of FACS analysis data.

FIGS. 5A-5B show photographs of plasma cells obtained from bone marrowaspirates of MM patients stained with H23 (FIG. 5A) or R23IgG (FIG. 5B).Stained cells are indicated by an arrow.

FIGS. 6A and 6B are a graphical representation of the percent of celllysis in a Complement dependent cytotoxicity (CDC) assay using therabbit polyclonal antibodies R23IgG (FIG. 6A) and the two monoclonalantibodies, SPmAb-2. I and SPmAb-6 (FIG. 6B) on various target celllines.

FIGS. 7A-7F are graphical representations of the levels of sMUC1 (MUC1Ag) as compared with the levels of endogenously generated antibodiesagainst VXL3A, VXL2S and VXLI00 in cancer patients having non-solidtumors (MM, FIGS. 7A-7C) and for cancer patients having solid tumors, inparticular Colon, Rectal, Lung, and Prostate (FIGS. 7D-7F).

FIGS. 8A-8D are graphical representations of the inter-correlation ofthe concentrations of various endogenously generated anti-MUC1 peptideantibodies in MM patients (FIGS. 8A and 8B) and in patients with solidtumors (FIGS. 8C and 8D).

FIGS. 9A-9C are graphical representations of the expression levels ofendogenously generated antibodies which recognize VXL25 (FIG. 9A),VXL100 (FIG. 9B) and VXL3A (FIG. 9C) in BRCA 1/2 (−), BRCAl (+) or BRCA2(+) carriers.

FIGS. 10A-10C show photographs of 4′-6-Diamidino-2-phenylindole (DAPI)DNA staining of MTb bacteria. FIG. 10D is a photograph showingimmunofluorescence staining of MTb bacteria with anti SP antibodies.FIG. 10E is a photograph showing immunofluorescence staining of arelated mycobacterium strain, M. Kansasii. FIG. 10F is a photographshowing immunofluorescence staining of MTb bacteria with sera fromnormal mice.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is based on the surprising finding that signalpeptide (SP) domains of disease-associated polypeptides are presented onthe cell surface of cells.

Moreover, the inventors demonstrate that antibodies directed against thesignal peptide (SP) domain of disease-associated polypeptides arecapable of detecting cell surface associated expression of these SPdomains in patients suffering from various diseases such as cancer orinfectious diseases, e.g. Mycobacterium tuberculosis.

The present invention therefore provides methods of diagnosing and/ortreating various diseases using antibodies directed against the signalpeptide (SP) domain of disease-associated polypeptides.

The present invention also provides methods of diagnosing diseases bydetermining the level of endogenous antibodies (also termed“autoantibodies”) which are produced by the patient and are directedagainst the signal peptide (SP) domain of disease-associatedpolypeptides.

Since the antibodies of the invention detect cell surface expression ofthe SP domain of a certain disease-associated polypeptide, they may beused as a tool for selecting patients suitable for treatment which isspecifically directed against that SP domain.

As a non limiting example, if a patient is found to express on the cellsurface of a tumor cell the SP domain of a disease associatedpolypeptide such as MUC1, such a patient is likely to benefit fromtherapy which is directed against MUC1 SP domain. Non limiting examplesof such therapy are a vaccine comprising the MUCl SP domain, e.g.ImMucin and/or antibodies which are directed against the MUC1 SP domain.Such antibodies may optionally be conjugated with cytotoxic moietieswhich facilitate cancer cell lysis.

Accordingly, by a first of its aspects, the present invention provides amethod for determining the suitability for treatment of a subjectsuffering from a disease, the method comprises:

-   -   a. contacting a biological sample containing cells obtained from        said subject with an antibody directed against a signal peptide        (SP), or any fragment thereof, of a polypeptide associated with        said disease; and    -   b. determining the expression level of said SP, or any fragment        thereof, on the surface of cells in said biological sample,

wherein the presence of said SP, or any fragment thereof, on the surfaceof said cells in a level higher than a control level indicates that saidsubject is suitable for treatment.

As used herein the term “treatment” refers to clinical intervention inan attempt 15 to alter the course of disease in the individual beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of the disease, alleviation ofsymptoms, reducing a pathological consequence of the disease, reducingthe rate of disease progression, amelioration of the disease state,remission or improved prognosis. The term “treatment” may also encompassex vivo procedures affecting cells or tissues in culture.

As used herein the term “subject” refers to an individual, or a patient,which is a vertebrate, e.g. a mammal, including especially a human.

As used herein the term “disease” refers to any condition whichameliorates the health of an individual. In one embodiment the presentinvention concerns a method for determining the suitability fortreatment of a subject suffering from cancer. In other embodiments thepresent invention concerns a method for determining the suitability fortreatment of a subject suffering from an infectious disease.

As used herein the term “cancer” refers to the physiological conditionin mammals that is typically characterized by unregulated cellgrowth/proliferation. The term cancer also refers to cancer cells.

As used herein, the term “polypeptide associated with a disease” or“disease associated polypeptide” refers to proteins whose expression ishighly correlated with the presence of certain diseases. These proteinsare not usually expressed in normal cells, or are expressed at a higherextent in diseased cells than in normal cells. Specific, non limitingexamples of disease associated polypeptides are, for example “TAA”.

As used herein, the term “tumor associated antigen” or “TAA” refers toantigens or proteins that are highly correlated with certain tumorcells. These antigens are not usually expressed in normal cells, or areexpressed at a higher extent in tumor cells than in normal cells. “TAA”also refers to cancer associated epitopes, cancer associated markerproteins, cancer associated proteins, or to a cancer marker. As usedherein, the term “tumor associated antigen fragment” refers to antigenicfragment that is recognizable by an antibody directed against said tumorassociated antigen.

Preferably, the disease associated polypeptide is expressed on the cellsurface and is amenable to recognition by elements of the immune systemsuch as immune cells or antibodies.

Specific, non limiting examples of TAAs include MUCI, Armet, HSP60,CANX, MTHFD2, FAP, MMP6, BAGE-1, GNTV, Q5H943, CEA, Pmel, Kallikrein-4,Mammaglobin-1, MART-I, GPR143-OA1, PSA, TRPI, Tyrosinase, FGP-5, NEUproto-oncogene, Aft, MMP-2, PSMA, Telomerase-associated protein-2, PAP,Uroplakin II and Proteinase 3.

In certain embodiments the present invention concerns a method fordetermining the suitability for treatment of a subject suffering from aMUC1 expressing cancer. As used herein the terms “MUCl”, “MUCJ antigen”,“MUCJ Ag”, or “MUCJ epitope” are used interchangeably and refer to ahigh molecular weight glycoprotein expressed on many cancer cell types.As used herein the term “MUCJ expressing cancer” refers to cancersexpressing MUC1, which may be, but are not limited to multiple myeloma(MM), breast cancer, ovary cancer, colon cancer, rectal cancer, gastriccancer, non-small lung carcinoma (NScLC), renal cell carcinoma (RCC),transitional cell carcinoma (TCC), prostate cancer, pancreatic cancer,thyroid cancer, Squamous cell carcinoma (SCC), Thymic Carcinoma,lymphoma, leukemia, and Mesothelioma.

Methods for determining whether a cancer type or a cancer cellover-expresses MUC1 are well known in the art, and generally includedetection of the MUC1 protein or mRNA encoding for the MUC1 protein.

As used herein the term “infectious disease” relates to a diseasemediated by an infectious agent. Such agents may include viruses,bacteria, parasites, prions and fungi. Hence, the term “polypeptideassociated with a disease” also encompasses polypeptides associated withinfectious agents causing an infectious disease.

In one specific embodiment the infectious agent is Mycobacteriumtuberculosis (MTb). In accordance with the invention, non limitingexamples of polypeptides associated with MTb include Antigen 85B,Lipoprotein IpqH, ATP dependent helicase putative, Uncharacterizedprotein Rv0476/MTO4941 precursor and Uncharacterized proteinRv1334/MT1376 precursor.

As used herein the term “biological sample” includes, but is not limitedto, plasma, serum, whole blood, urine, sweat, lymph, faeces,cerebrospinal fluid, bone marrow biopsy or aspirate, nipple aspirationor other biological sample derived from a subject.

As used herein the term “signal peptide” (SP) refers to a short (15-60amino acids long) peptide chain that directs the transport of a protein.Signal peptides are also referred to as “targeting signals”, “signalsequences”, “transit peptides”, or “localization signals”. The inventionencompasses both SP expressed on the cell surface of cells or to solubleSP, as well as to peptide fragments thereof.

A ‘fragment of an SP’ as used herein is defined as any peptide fragmentof a SP which is recognizable by a binding agent, e.g. an antibody. Incertain embodiments, for epitope recognition in the context of MHC classI, said fragment may be 9 amino acids long and for MHC class II, 15amino acids long. The epitopes may be linear or conformational epitopes.In certain specific embodiments, the fragment comprises the C-terminusof the SP. The “C-terminus” (also known as the carboxyl-terminus,carboxy-terminus, C-terminal tail, C-terminal end, or COOR-terminus)refers to the end of the SP amino acid chain terminated by a freecarboxyl group (—COOH), and may comprise one or more amino acids at theC-terminus. In certain embodiments an amide is used at the C terminus.

Identifying SP sequences for a particular disease-associated polypeptidemay be performed by using any appropriate method known in the art. Inparticular, SP sequences may be identified using computer software, e.g.the signal P 3.0. The signal P 3.0 program

30 uses both a neural network (NN) algorithm and a hidden Markov Models(HMM) algorithm for selection of the signal sequence. In certainembodiments, the epitopes in accordance with the invention may be anincomplete SP. For example, without wishing to be bound by theory, theC-terminal of the SP may be more relevant for cellular interaction as itis probably the part that gets to the surface without the MHC. Asequence is considered to be a SP whenever a score of over 0.2 wasreceived in one or more of the algorithms. Sequences having a score ofabove 0.7 are preferred. Sequences having a score of above 0.8 are mostpreferred.

Examples of SP sequences are provided in Table I below, and includeVXLI00 which consists of the entire MUC1 SP domain, and shorter peptideswhich correspond to MUC1 SP fragments, e.g. VXL3A, VXLI, VXL2, VXL3,VXL4, VXL5, VXL13 and VXL15. The table also shows the SP of the TAAsArmet (VXLI0I), BAGE (VXL102), Uroplakin II (VXL104), PAP (VXL106) andMammaglobin-1 (VXL108), as well as the SP sequences of several MTbproteins including Ag85B (VXL201), Lipoprotein lpqH (VXL203), ATPdependent helicase putative protein (VXL208), Uncharacterized proteinRv0476/MTO4941 precursor (VXL21 1), and Uncharacterized proteinRv1334/MT1376 precursor (VXL212).

TABLE 1 List of sequences SEQ ID NO. Sequence Name Description  1STAPPAHGVTSAPDTRPAPGST VXL25, BP25 or A peptide derived from the APPMUC1-TRA-L TRA domain of human MUC 1.  2 MTPGTQSPFFLLLLLTVLTVV VXL100 orA peptide consisting of the entire MUC1-SP-L domain of human MUC1 SP.VXL100 is the antigenic portion (API) of the vaccine ImMucin.  3KKFLLLLLTVLTVVKKK VXL3A or A peptide which consists of the MUC1-SP-Mfragment 10-21 of human MUC1 SP and includes five additionallysines at the N- and C- terminal.  4 LLLTVLTVV VXL1, MUC1-A peptide which consists of the SP-S1 or fragment 13-21 in human MUC1D6MUC1 SP.  5 LLLLTVLTV VXL2, MUC1- A peptide which consists of theSP-S2 or fragment 12-20 in human MUC1C6 MUC1. SP.  6 FLLLLLTVL VXL3 orA peptide which consists of the MUC1-SP-S3 fragment 10-18 of human MUC1SP.  7 TQSPFFLLL VXL4 or A peptide which consists of the MUC1-SP-S4fragment 5-13 in human MUC1 SP.  8 SPFFLLLLL VXLS orA peptide which consists of the MUC1-SP-SS fragment 7-15 in human MUC1SP.  9 FFLLLLLTV VXL13 A peptide which consists of thefragment 9-17 in human MUC1 SP. 10 MTPGTQSPF VXL15A peptide which consists of the fragment 1-9 in human MUC1 SP. 11MWATQGLAVALALSVLPGSR VXL101 A peptide which consists of the Aentire SP domain of the human protein Armet. 12 MAARAVFLALSAQLLQAVXL102 or A peptide which consists of the BAGE-SP-Lentire SP domain of the human protein BAGE. 13 MAPLLPIRTL PLILILLALLVXL104 A peptide which consists of SPGAA the entire SP domain of thehuman TAA Uroplakin II. 14 MFDKTRLPYVALDVLCVLLAG VXL106A peptide which consists of the LPFAIL entire SP domain of the humanTAA PAP. 15 MKLLMVLMLAALSQHCYA VXL108 A peptide which consists ofthe entire SP domain of the human TAA Mammaglobin- 1. 16MTDVSRKIRAWGRRLMIGTAA VXL201 A peptide which consists of theAVVL PGLVGLAGGAATAGA entire SP domain of the Mycobacterium tuberculosisprotein Ag85B. 17 MKRGLTVAVAGAAILVAGLSG VXL203A peptide which consists of the CSS entire SP domain of theMycobacterium tuberculosis protein Lipoprotein 1pqH. 18MRFAQPSALSRFSALTRDWFTST VXL208 A peptide which consists of theFAA PTAAQA entire SP domain of the Mycobacterium tuberculosisATP dependent helicase putative protein. 19 MLVLLVAVLVTAVYAFVHAVXL211 or TB- A peptide which consists of Rv0476/4941-the entire SP domain of the SP-L Mycobacterium tuberculosisprotein Uncharacterized Rv0476/MTO4941 precursors. 20MLLRKGTVYVLVIRADLVNAM VXL212 A peptide which consists of VAHAthe entire SP domain of the Mycobacterium tuberculosisprotein uncharacterized Rv1334/1376.

In accordance with the invention the SP domain of a desired diseaseassociated polypeptide is used for the preparation of specificantibodies, i.e. anti SP antibodies. Such antibodies may be used in themethod for determining the suitability for treatment of a subjectsuffering from a disease as well as in additional aspects of theinvention as would be discussed below.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and specifically refer to a polyclonal antibody, amonoclonal antibody, or any fragment thereof, which retains the bindingactivity of the antibody. In certain embodiments the use of a chimericantibody, a humanized antibody, or a human antibody is also encompassedby the invention.

As used herein the term “polyclonal antibody (or antibodies)” refers toa population of different antibodies directed against differentdeterminants (epitopes) of the same antigen.

The term “monoclonal antibody (or antibodies)” as used herein refers toa population of substantially homogenous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossibly naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are directed against a single antigenicsite.

The anti SP monoclonal antibodies of the invention can be made using thehybridoma method first described by Kohler et al, Nature, 256: 495(1975), or may be made by recombinant DNA methods (e.g. U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to the SP generally are raisedin animals by subcutaneous (sb) or intraperitoneal (ip) injections ofthe desired SP (for example SP domains listed in Table 1) and anadjuvant. In one embodiment, the animals are immunized with the SPcoupled to Keyhole limpet hemocyanin (KLH, Sigma Aldrich) as a carrierprotein.

The signal peptides used for animal immunization are prepared usingmethods well-known in the art. For example, the SP may be produced byrecombinant methods or by peptide synthesis methods.

Alternatively, lymphocytes may be immunized in vitro and then fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal Biochem.,107: 220 (1980).

The anti SP antibodies of the invention can be made by usingcombinatorial libraries to screen for synthetic antibody clones with thedesired activity. In principle, synthetic antibody clones are selectedby screening phage libraries containing phage that display variousfragments of antibody variable region (Fv) fused to phage coat proteinusing methods well known in the art.

As used herein the term “any fragment thereof which retains the bindingactivity of the antibody” refers to a portion of an antibody, preferablycomprising the antigen-binding or variable region thereof, which iscapable of binding to the target antigen of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments.

These antibody fragments may be generated by recombinant techniques orby traditional means, such as enzymatic digestion. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single binding site, and a residual “Fe”fragment. Pepsin treatment yields an F(ab′)2 fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site.

The polyclonal antibodies and the monoclonal antibodies of the inventionare prepared using methods well known in the art.

A specific non limiting example of methods for preparing anti SPantibodies is provided in Example 1 below.

In specific embodiments, the antibodies used in the methods of theinvention include the polyclonal preparations denoted R23 or R23IgG(which is an IgG fraction of rabbit's polyclonal hyper immune seradirected against the 17 amino acid long MUCl SP peptide VXL3A (SEQ IDNO: 3)) or SPimAb-3A (which is an IgG fraction of rabbit's polyclonalhyper immune sera which is directed against the 21 amino acid long MUC1SP peptide VXL100 (SEQ ID NO: 2)). In other embodiments the antibodiesused in the methods of the invention include the monoclonal antibodiesdenoted SPmAb-2.1 and SPmAb-6. These monoclonal antibodies are directedagainst the 17 amino acid long MUCl SP peptide VXL3A (SEQ ID NO: 3). Thehybridoma cell producing the SPmAb-6 antibody was deposited at the

ECACC on Sep. 13, 2012 and received Accession no. 12091301.

Interestingly, the polyclonal antibodies directed to the 1 7mer (17amino acid long peptide) MUC1 SP were found to be more specific to theMUC1 SP Domain as compared with the polyclonal antibodies directed tothe 21mer (21 amino acid long peptide) MUCl SP.

The cell surface expression level of the SP of the disease associatedpolypeptide may be determined using the anti SP antibodies in anysuitable detection method known in the art, for example, by employingELISA, RIA, FACS analysis or immunohistochemistry.

The level of expression is measured and compared with the level ofexpression of a suitable control. A control may be the level ofexpression of a non-relevant SP (e.g. of a non-relevant TAA, or of aforeign antigen (of an infectious agent) such as an MTb), or the levelof expression of the same SP on a normal, non diseased cell of the sameor a different individual.

In one embodiment, “positive” is defined as having at least a doublegeometric mean of the control. In one embodiment, “negative” result inFACS analysis is defined as absence or <30% increase of the geometricmean in a sample stained with the evaluated Ab as compared with a samplestained with isotype control (e.g. normal mouse or rabbit polyclonal ormonoclonal Abs FITC conjugated).

A subject is found to be suitable for treatment whereby cell surfaceexpression of the SP (or the SP fragment) of the disease-associatedpolypeptide is detected.

In specific embodiments the treatment encompasses administering an agentwhich is specifically directed against the SP of the disease-associatedpolypeptide, i.e. an agent capable of binding to the SP. The term “anagent capable of binding” as used herein refers to an agent capable ofbinding an antigen with sufficient affinity such that the agent isuseful as a therapeutic agent in targeting a cell expressing theantigen. Preferably, the agent is capable of causing (directly orindirectly) the destruction of cells harboring these SP domains.

Such agents may be, but are not limited to, a ligand, an antibody, acombination of antibodies, or any fragment thereof, capable of bindingthe SP domains. In one embodiment, binding of antibodies to cells viathe SP domains may result in the selective elimination of such cells bythe complement system.

In other specific embodiments the treatment encompasses administering avaccine capable of eliciting an immune response against the SP, or anyfragment thereof, thereby inducing the immune system of the subject toraise or enhance an immune response against the cells harboring the SPdomains. As used herein, the term “vaccine” refers to a composition thatimproves immunity to a particular disease.

Examples of such vaccines may be found in WO 2008/035350 which relatesto SP-derived vaccines, capable of inducing a robust, antigen specificT-cell immunity and which are applicable to the majority of thepopulation.

In one specific embodiment the vaccine comprises a MUC1 SP, e.g. VXLI00or VXL3A, or a formulated version of the MUC1 SP, known as ImMucin. Inother embodiments the treatment may include a combination of a MUC1 SPvaccine (e.g. ImMucin, VXLI00 or VXL3A) and at least one anti MUC1 SPantibody.

Without wishing to be bound by theory, one advantage of such treatmentsis the relative low toxicity thereof, since these treatments arespecifically directed to cells presenting the SP domains on their cellsurface.

The treatment may include a combination of a vaccine and at least oneantibody, or a combination of several antibodies with and without avaccine. Specifically, the

10 invention encompasses a therapeutic regime including a combination ofa MUC1 SP vaccine (e.g. ImMucin) and anti MUC1 SP antibodies. Theinvention also encompasses a therapeutic regime including a combinationof anti MUC1 SP antibodies together with additional agonistic antibodiesdirected against MUC1 or any other suitable TAA.

Since the antibodies of the invention detect cell surface but notsoluble expression of the SP domain of a certain disease-associatedpolypeptide, they may be used as a therapeutic tool for selectivelydestroying cells which express these SP domains on their cell surface.

This effect may be mediated by activation of the complement system or byattaching a cytotoxic moiety to the anti SP antibodies.

As a non limiting example, antibodies directed against MUC1 SP domainmay be used for causing cell death of MUC1 expressing cancer cells or ina method of treating MUC1 expressing cancers. The antibodies mayoptionally be conjugated with cytotoxic moieties which facilitate cancercell lysis.

Accordingly, by a second of its aspects, the present invention providesa method of treatment of a subject suffering from a disease, the methodcomprises administering to said subject a therapeutically effectiveamount of at least one antibody directed against a signal peptide (SP),or any fragment thereof, of a polypeptide associated with said disease,whereby the binding of said antibody to cell surface expressed SPdirectly or indirectly results in cell death.

The invention also provides a method of inducing cell death orinhibiting cell growth, comprising administering to a population ofcells at least one antibody directed against a signal peptide (SP), orany fragment thereof, of a polypeptide associated with said disease,whereby the binding of said antibody to cell surface expressed SPdirectly or indirectly results in cell death.

Optionally, the antibody is administered in combination or inassociation with a cytotoxic moiety.

As used herein the term “cytotoxic moiety” refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term includes, for example, a radioactive agent, a toxin, ananti-metabolite, or an alkylating agent. The conjugation of the antibodywith the cytotoxic moiety is performed using methods well known in theart.

The invention therefore also provides use of immune conjugates of theanti SP antibodies (i.e. antibody-drug conjugates), comprising any ofthe anti SP antibodies described herein conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, a drug, a growth inhibitory agent, atoxin (e.g. an enzymatic active toxin of bacterial, fungal, plant oranimal origin, or fragments thereof) or a radioactive isotope.

Optionally, the antibody mediates cell lysis via complement activation.

The term “a therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic or prophylactic result.

As used herein the term “cell death” refers to a viable cell whichbecomes nonviable, cell death may be caused by lysis, apoptosis orindirectly as a result of inhibition of cell proliferation or celldivision (encompassing both a cytolytic and a cytostatic effect).

As used herein, the anti SP antibodies may be monoclonal or polyclonalantibodies as described above. In specific embodiments, the antibodiesare non human antibodies e.g. mouse or rabbit antibodies. In otherembodiments the anti SP antibodies are chimeric antibodies, or humanizedantibodies or human antibodies.

As used herein the term “chimeric antibody” refers to antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular

30 antibody class or subclass, while the remaining chain(s) areidentical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass. For example, a chimeric antibody may include a human Feportion and a mouse or rabbit variable region.

As used herein the term “humanized antibody” refers to antibodies whichhave a human backbone and contain minimal sequences (e.g. in thecomplementarity determining region, CDR) derived from non-humanimmunoglobulin.

A “human antibody” is an antibody which comprises an amino acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies known tothose skilled in the art.

In one non limiting example, an amount of between 4 and 1 6 mg/Kg bodyweight of anti MUC1 SP antibodies (e.g. anti-VXLI00 antibodies) areadministered to an individual (for example by intra peritoneal, or intravenous injection) 1, 2 or 3 times at a weekly interval. The exact dosageform and regimen would be determined by the physician according to thepatient's condition.

Since the antibodies of the invention detect cell surface expression ofthe SP domain of a certain disease-associated polypeptide, they may beused as a tool for diagnosing patients suffering from the disease. Insuch case the SP expression serves as a disease marker.

As a non limiting example, a biological sample obtained from anindividual may be screened for cell surface expression of MUC1 SP.Expression of MUC1 SP may be indicative of disease, e.g. multiplemyeloma. Preferably, the screening is performed with a combination ofanti MUC I SP antibodies with detectors of additional cell markers.

Accordingly, by another aspect, the present invention provides a methodof diagnosing a disease in a subject, the method comprises:

-   -   a. contacting a biological sample containing cells obtained from        said subject with an antibody directed against a signal peptide        (SP), or any fragment thereof, of a polypeptide associated with        said disease; and    -   b. determining the level of said SP, or any fragment thereof, on        the surface of cells in said biological sample,

wherein the presence of a level of said SP in said sample which ishigher than a control level is indicative of a disease.

The present invention also provides methods of diagnosing diseases bydetermining the level of endogenous antibodies (also termed“autoantibodies”) which are produced by the patient and are directedagainst the signal peptide (SP) domain of disease-associatedpolypeptides.

Accordingly, by yet another aspect, the present invention provides amethod for detecting a disease in a subject, said method comprises:

-   -   a. contacting a biological sample obtained from said subject        with at least one SP, or any fragment thereof, of polypeptide        associated with said disease;        -   and    -   b. measuring the level of endogenous antibodies directed against        said SP, or any fragment thereof, in said biological sample,

wherein the presence of said endogenous antibodies in the sample in alevel higher than a control is indicative of disease

As used herein the term “endogenous antibodies” refers to antibodiesgenerated in a subject's body by its own immune system.

In accordance with this aspect of the invention the level of endogenousanti SP antibodies in a biological sample is determined by contactingthe sample with a SP or a fragment thereof.

Methods for identifying SP domain sequences for use in accordance withthe invention, were described above. Peptides based on these SP domainsmay be used for detecting anti SP endogenous antibodies present in thebiological sample.

The nomenclature used to describe peptide compounds of the inventionfollows the conventional practice wherein the amino group (N-terminus)is presented to the left and the carboxyl terminus (C-terminus) ispresented to the right.

Derivative of the peptides are also included in the present invention.Derivatives are meant to include peptides which differ in one or moreamino acids in the overall sequence, which have deletions,substitutions, inversions or additions. It is appreciated that thesepeptide modifications and peptide derivatives must not alter thestructure of the original peptides in a manner that abrogates theability of endogenous antibodies to recognize and bind these modifiedpeptides.

The peptides according to the invention can be produced synthetically,or by recombinant DNA technology. Methods for producing peptides arewell known in the art.

The level of binding of endogenous antibodies to the SP domains may beperformed using any immunological technique known in the art.Particularly, the level of endogenous antibodies may be measured usingELISA, radioimmunoassay, or similar techniques.

Examples of methods for detecting anti SP endogenous antibodies areprovided in the Examples below.

In a specific embodiment, the present invention provides a method ofquantifying anti MUC1 SP autoantibodies using ELISA assays with MUC1 SPspecific epitopes in

5 the sera of multiple myeloma cancer patients. Interestingly, the levelof such antibodies is significantly increased in MM patients as comparedto healthy individuals thereby providing a tool for diagnosing thedisease. Without wishing to be bound by theory, this significantincrease stems chiefly from the preferred immunogenicity of the signalpeptide.

Naturally generated autoantibodies to TAAs are detectable even beforethe tumor is clinically apparent (Lu H. et al J. Proteome Res. (2008) 7:1388-1394), and due to their lower fluctuation and longer half-life inthe blood, they may be more appropriate for cancer diagnosis thanautoantibodies directed to non-SP domains of TAAs.

The present invention also provides specific antibodies directed to SPdomains of peptides associated with a disease, or fragments thereof, foruse in the above described methods for example to identify subjects thatmay benefit from or may be suitable to treatment as defined above.

Importantly, the antibodies of the invention may also be specificallyused for treatment of subjects suffering from a disease. As a nonlimiting example, the antibody of the invention may be a neutralizingantibody, or an antibody associated with or combined with a cytotoxicmoiety.

Therefore, in yet another aspect, the present invention provides anisolated antibody directed against a signal peptide (SP), or anyfragment thereof, of a polypeptide associated with a disease, for use ina method of determining the suitability for treatment of a subjectsuffering from said disease, wherein said method comprises:

-   -   c. contacting a biological sample containing cells obtained from        said subject with said isolated antibody; and    -   d. determining the expression level of said SP, or any fragment        thereof, on the surface of cells in said biological sample,        wherein the presence of said SP, or any fragment thereof, on the        surface of said cells in a level higher than a predetermined        baseline indicates that said subject is suitable for treatment.

In yet another aspect, the present invention provides an isolatedantibody directed against a signal peptide (SP), or any fragmentthereof, of a polypeptide associated with a disease, for use in a methodof treatment of a subject suffering from said disease, wherein saidmethod comprises administering a therapeutically effective amount ofsaid antibody to the subject.

In yet another aspect, the present invention provides an isolatedantibody directed against a signal peptide (SP), or any fragmentthereof, of a polypeptide associated with a disease for use in a methodof diagnosing a disease in a subject, said method comprises:

-   -   C. contacting a biological sample containing cells obtained from        said subject with said isolated antibody; and    -   d. determining the level of said SP, or any fragment thereof, on        the surface of cells in said biological sample,        wherein the presence of a level of said SP in said sample which        1s higher than a predetermined baseline level is indicative of a        disease.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.

EXAMPLES Example 1 Generation of Antibodies Directed Against the SPDomain of MUC1

Animals used for producing antibodies were six to 8-week-old femaleBALB/c mice and two month old rabbits (the Tel-Aviv university breedingfacility). Animals were maintained in the university animal researchfacility. All experiments were conducted according to the Tel-Avivuniversity institutional rules and regulations.

The peptides MUCl-SP-L (also referred to as VXLlO0 or denoted by SEQ IDNO.2), MUCl-SP-M (also referred to as VXL3A or denoted by SEQ ID NO.3),MUCl-SP-S1 (also referred to as VXL1, MUC1D6 or denoted by SEQ ID NO.4),MUC1-SP-S2 (also referred to as VXL2, MUC1C6 or denoted by SEQ ID NO.5),MUC1-SP-S3 (also referred to as VXL3 or denoted by SEQ ID NO.6),MUC1-SP-S4 (also referred to as VXL4 or denoted by SEQ ID NO.7),MUC1-SP-S5 (also referred to as VXL5, or denoted by SEQ ID NO.8) andTB-Rv0476/4941-SP-L (also referred to as VXL211, or denoted by SEQ IDNO.19) were synthesized by fully automated, solid-phase, peptidesynthesis using fluorenylmethyloxycarbonyl (Fmoc)/tBu-strategy andRink-amide-polystyrene resin at EMC Microcollections, Germany, whileMUCl-TRA-L (also referred to as VXL25) and BAGE-SP-L were synthesizedusing the same methodology at GL Biochem, China. The purity and identityof all peptides was >95%, as determined by HPLC and MS analysis.Nomenclature used in selected peptide is as follows: “S” denotes short,“M” denotes moderate and “L” denotes long.

A 17mer SP domain of MUC1 (MUCI-SP-M peptide, also denoted by SEQ ID NO.3, or VXL3A, Table 1) coupled to Keyhole limpet hemocyanin (KLH, SigmaAldrich) as a earner protein was used for generating polyclonal andmonoclonal antibodies. The MUCl-SP-M peptide-KLH conjugation wasprepared by cross linking with Glutaraldehyde by Adar biotech (Rehovot,Israel), according to methods well known in the art. MUCl-SP-Mconjugated to KLH was emulsified with complete Freund's adjuvant in thefirst immunization and Incomplete Freund's adjuvant in subsequentimmunizations.

Additional polyclonal antibodies were also generated against the 21merpeptide derived from MUCl SP (i.e., VXL100, also denoted by SEQ ID NO.2) using the same methods as described for the 17mer antigen, and termedSPimAb-3A.

Anti MUC1 SP Polyclonal Antibodies

Polyclonal antibodies were prepared as follows: Four 2 months oldrabbits were subcutaneously immunized five times at weekly intervals.Samples were obtained from the immunized rabbits after each of the firstthree immunizations in order to check the titer quality. Then, twoadditional injections were administered at weekly intervals and therabbits were boosted when a decline in the antibodies titer wasobserved.

After the final immunization (at day 58), rabbits sera were examined forthe presence of specific antibodies directed against MUCl-SP-M (alsotermed VXL3A or denoted by SEQ ID NO. 3), and positive sera (titer1:12,500) were collected and pulled.

20 For all immunological assays, an IgG fraction was used, following 40%ammonium sulfate precipitation as previously described in Alausa, O. K.[Journal of immunological methods. 8(1-2):117-126 (1975)].

Polyclonal Antibody Specificity

Titers of anti-MUC1-SP-M polyclonal antibody sera of up to 1:12,800dilutions were obtained in two of the immunized rabbits, namely R23 andR32 (FIG. 1AI). The polyclonal antibody preparations were accordinglydenoted R23 and R32. In these experiments, an optical density (O.D.)larger than 0.1 (O.D.>0.1) was considered as a positive result. Thespecificity of the antibodies directed to MUC1 was high and showedlimited cross reactivity with other SP domains (titers of <1:800), asshown in FIGS. 1AII and 1AIII: FIG. 1AII demonstrates a limited crossreactivity observed between the antibodies directed to MUCl and thepeptide BAGE-SP-L (also denoted by SEQ ID NO. 12, Table 1), which isderived from the eukaryotic protein BAGE, and FIG. 1AIII demonstrates alimited cross reactivity observed between the antibodies directed toMUCI and the peptide TB-Rv0476/4941-SP-L (also denoted by SEQ ID NO. 19,Table 1), derived from the bacteriaAfycobacterium tuberculosis.

As demonstrated in FIG. 1B, the inner epitopes of MUCI-SP-M which werehighly recognized by the R23 antibodies were MUCI-SP-Sl (also denoted bySEQ ID NO. 4, Table 1) and MUC1-SP-S2 (also denoted by SEQ ID NO. 5,Table 1). These peptides are located at the C-terminus of MUCl SP.

As demonstrated in FIG. 1C, over 50% inhibition was observed both forR23 and for R32 polyclonal antibodies by MUCI-SP-M (also denoted by SEQID NO. 3) and its inner epitope MUC1-SP-S2 (also denoted by SEQ ID NO.5). However, less than 10% inhibition was achieved by other MUC I SPepitopes, in particular MUC 1-SP-S4 (also denoted by SEQ ID NO. 7) andthe MUCI TRA epitope MUCI-TRA-L (also denoted by SEQ ID NO. 1) or by theBAGE SP domain BAGE-SP-L (also denoted by SEQ ID NO. 12).

Anti MUC I SP Monoclonal Antibodies

Monoclonal antibodies were prepared as follows: Four BALB/c mice weresubcutaneously immunized four times (at weekly intervals) with theMUCl-SP-M (also named VXL3A, or denoted by SEQ ID NO.3) peptide (100 μg)conjugated to KLH and emulsified in Complete Freund's adjuvant (CFA) atthe first immunization and in Incomplete Freund's adjuvant (IFA) at thesubsequent immunizations. After the final immunization step (two weeks),mice sera were examined for the presence of specific antibodies againstMUCl-SP-M. Titers of anti-MUCl-SP-M polyclonal antibody sera of up to1:25,000 dilutions were obtained in the immunized mice. Spleen cellsobtained from the mouse bearing the highest positive sera were harvestedand fused with the murine myeloma partner NSO cell line, usingpolyethylene glycol (molecular weight 1500, Roche Diagnostics GmbH,Germany). Hybridomas cells were selected for two weeks in DMEM mediasupplemented with 10% of horse serum, L-Glutamine, Sodium Pymvate,Gentamycin and Hypoxanthine, Aminopterin, Thymidine (HAT) mixture (BeitHaemek IL) and were further cultured for additional two weeks in asimilar growth medium with Hypoxanthine, Thymidine (HT) mixture in 96F Wplates (DeGroot Grainer Germany). Culture supernatants from each wellwere screened for the presence of anti-MUCl-SP-M IgG antibodies (Abs) byELISA. Hybridoma cells from wells producing Abs were isolated retestedand subjected to sub-cloning. Large-scale Abs production of selectedclones was achieved by purifying monoclonal antibodies (mAbs) fromculture media using anti-Mouse IgG agarose column (Cat. No A653 1,Sigma, Israel). Isotyping of mAbs was performed using the Isostrip kit(Roche Cat. no. 1493027).

Monoclonal Antibody Specificity

Binding experiments performed in mouse No. 1 (Ml), as presented in FIG.1D, showed strong binding of >1:12,800 titer against the immunizingpeptide MUC1-SP-M, moderate binding of >I:1800-3600 titers to peptidesMUC1-SP-Sl, MUC1-SP-S2 and MUC1-SP-S3 (denoted by SEQ ID NO. 4, 5 and 6,respectively) and low binding >1:800 titer to peptides MUC1-SP-S4 andMUC1-SP-S5 (denoted by SEQ ID NO. 7 and 8, respectively). No binding wasobserved with the MUC1-TRA-L peptide.

Hybridoma formation resulted in 2 monoclonal antibodies, namelySPmAb-2.1, originated from mouse No. I and having an Ig-gamma1 isotype,and SPmAb-6, originating from mouse No. 2 and having an Ig-gamma2aisotype. The specificity of the two generated mAbs was validated byperforming binding and competition assays as those detailed above (FIG.1E) in the presence of the various free soluble peptides indicatedtherein. As demonstrated in FIG. 1E, the peptides MUC1-SP-M, MUC1-SP-SIand MUCl-SP-S2 present about 60%, 50% and 80% inhibition, respectively,with respect to the mAb SPmAb-2.1, while the peptides MUCl-SP-S4 andMUC1-SP-SS both showed around 30% inhibition with respect to the samemAb. Interestingly, the peptides MUC1-SP-S4 and MUC1-SP-S5, manifestedabout 80% and 65% inhibition, respectively, of mAb SPmAb-6, while thepeptides MUC1-SP-M, MUC1-SP-Sl and MUC1-SP-S2, presented about 63%, 25%and 20% inhibition, respectively, of the same mAb. No inhibition wasmanifested by the MUC1-TRA-L peptide, for both mAbs. Based on theseresults the minimal epitope of SPmAb-2.1 is located within the sequenceof MUC l-SP-S2 peptide (denoted by SEQ ID NO. 5) and the minimal epitopeof SPmAb-6 is located within the sequence of MUCl-SP-S4 peptide (denotedby SEQ ID NO. 7).

Antibody Screening Assay

Screening of rabbits hyperimmune sera, mice hyperimmune sera andhybridoma producing anti-MUCl-SP-M IgG mAbs was performed using an ELISAprotocol, as follows: 96 well ELISA plates (F96 Maxisorp, Nunc, Denmark)were activated for 1 h with 0.1% of Glutaraldehyde (Sigma, IL) incarbonate buffer (pH=9). Plates were then coated with 50 μl of theevaluated peptide (as detailed above), at 5 μg/ml, in carbonate buffer,for overnight incubation at 4° C. followed by blocking for 2 h at roomtemperature with a blocking buffer consisting of PBS supplemented with5% FBS and 0.04% Tween 20 (ICN Biomedical Inc, USA). Evaluated serasamples from MUCl-SP-M immunized animals were then diluted 1:100 plus 7additional dilutions in PBS supplemented with 5% FBS and 0.04% Tween 20.Hybridoma growth medium were used (without dilution) and incubated for 2h at room temperature. Next, 50 μ1/well of the secondary anti-mouse oranti-rabbit IgG antibody HRP-conjugate (Jackson ImmunoResearch, USA) wasadded, at a final dilution of 1:10,000 in a blocking buffer andincubated for 1 h at room temperature. Plates were then developed withTMB/E solution (3,3′,5,5′-tetramethylbenzidine, CHEMICON, Millipore,USA) according to manufacture instructions.

For peptide antibody competition assays, rabbit or mice hyperimmune seraand Hybridoma growth medium were incubated together with I μg/w ofdifferent peptides on

35 96 well ELISA plates (F96 Maxisorp, Nunc, Denmark) activated withGlutaraldehyde as described above. The competition assay was performedas described for the ELISA above.

Example 2 Antibodies Recognize MUC1 SP on the Cell Surface of PreloadedAPC

Cell surface expression of an antigen is an important advantage for itsuse as a target for antibody recognition. Such cell surface expressionand antibody recognition can be employed for various diagnostic andtherapeutic purposes.

In the following example it is demonstrated that the antibodies of theinvention are capable of recognizing MUC1 SP expressed on the surface ofpreloaded antigen presenting cells (APC).

An anti MUC1 SP polyclonal antibody preparation, SPimAb-3A was used toevaluate the expression of MUC1 SP on the cell surface of cells. Thisevaluation was performed using Fluorescence-activated cell sorting(FACS) analysis.

SPimAb-3A staining of naïve macrophages (expressing both MHC class I andII molecules), which were preloaded with the VXLI00 peptide or a controlpeptide (such as VXLI0I and VXL102 which are the SP domains of non-MUC1TAA ARMET and BAGE, respectfully) was analyzed using FACS. I 00μ1containing cells at a concentration of 10×10⁷ cells/ml were incubatedfor 30 min at RT in FACS staining buffer, consisting of PBS supplementedwith 3% FCS, 0.1% sodium azide, and 10% Human AB serum (Sigma Israel,Rehovot, Israel). Then, the cells were transferred into FACS tubes (BDFalcon™, Franklin Lakes N.J. USA) and the staining buffer was carefullyremoved. For the staining step, 30μ1 of FITC-conjugated (Sigma Israel,Rehovot, Israel) H23, or SPimAb-3A in staining buffer (without AB serum)were added for an incubation of 30 min at RT, in the dark. Followingthis incubation step, cells were washed with 3 ml of staining buffer,and re-suspended in 0.5 ml PBS. Samples were then analyzed in a LSR IIFACS (BD Biosciences, San Jose, Calif., USA). The same macrophages,unloaded, or the anti-MUC1 TRA, or mAb H23 antibodies were used ascontrols.

H23 is monoclonal antibody that was raised against the human breastcancer cell line T47D and recognizes the epitope APDTRP on thenon-glycosylated form of MUCl's TRA (Keydar et al Proc. Natl. Acad. Sci.USA 1989, 86:1362-1366). MAb H23 recognizes the soluble MUCl's TRAdomain in sera or on cancer cells. It also recognizes the 25mer peptideVXL25.

The Results, demonstrated in Table 2 below, revealed 30% specificbinding by SPimAb-3A antibodies to VXLI00-loaded macrophages, comparedto no binding of SPimAb-3A antibodies to unloaded naïve macrophages orto macrophages loaded with control peptides (VXLI0I, VXL102). Theabsence of binding to un-loaded macrophages suggests that SPimAb-3Aantibodies specifically recognize the MUC1 epitope, which is notpresented on naive primary macrophages.

TABLE 2 Cells Expression(%) Naïve Macrophage Species H23 SPlmAb-3A Unloaded Human 13% 0 Loaded with VXL100 Human 12%  30% Loaded with VXL101Human 44% 0 Loaded with VXL102 Human 26% 4.8%

In addition, the results presented in Table 2 demonstrate a highernon-specific binding observed for H23 antibodies (at the range of10-40%), suggesting this antibody is of a lower specificity for theassayed peptides. The high specific binding demonstrated for SPimAb-3Aantibodies, particularly in view of the fact that SPimAb-3A is apolyclonal antibody, wherein H23 is a monoclonal antibody, isfundamental in confirming that SPimAb-3A has both a high MUC1 specificrecognition and a lower background binding properties to naïvemacrophages, in comparison to H23.

Example 3 Anti-MUC1 SP Antibodies Bind to MUC1-Positive Tumor Cells

In the following example it is demonstrated that the antibodies of theinvention are capable of recognizing cell surface expressed MUC1 SP onvarious types of cells.

An enriched IgG fraction of the rabbit R23 hyper immune sera(polyclonal) R23IgG, the IgG purified monoclonal antibodies SPmAb-2.1and SPmAb-6 and the H23 antibodies were used to determine the expressionprofile of MUC1 SP and other MUC1 epitopes on various human tumor cells(primary cells and cell lines). Binding of the antibodies to theepitopes was measured using FACS analysis and the results are presentedas percent or geometric mean of positive binding to cells, related tothe species-specific Isotype control. The FACS analysis was performed asdescribed above.

Flow cytometric analysis demonstrated that SPmAb-2.1, SPmAb-6 and R23IgGhave respectively moderate to high binding to MUC1 expressing tumorcells, both in solid tumors e.g. OVCAR-3 Ovarian carcinoma tumor, and innon-solid cancer cells e.g. RPMI, U266 MM cells, Raji and RamosB-Lymphocytic Leukemia tumors and ARH77 plasma cell Leukemia (Table 3).The monoclonal antibody directed against MUC1 TRA, namely, H23 [KeydarI, et al. PNAS USA 86:1362-1366 (1989)] was used as a positive controlfor MUC1 alfa-subunit (the part of MUCl containing the TRA domain whichis cleaved and released to the serum) and showed reactivity with similarbinding strength to the IgG fraction of the R23 antibodies. In contrast,MUC1 negative melanoma cell-lines SK-mel-28, SK-mel-1 and MDCI-negativeovarian cell-line ES-2 were consistently negative with all antibodies(Table 3) supporting the selective binding to MUC1. The nature andlocation of the antigen recognized by the different MUC1 SP antibodiesvs. the control MUC1 TRA antibody H23, was further characterized byImmunofluorescence microscopy staining. No staining of ES-2 ovariancarcinoma cells was seen with mAb H23, IgG fraction of the polyclonalantibodies R23 and mAbs SPmAb-2.1 and SPmAb-6 (FIG. 2A). However,membrane staining of OVACAR-3 ovarian carcinoma as well as MM RPMI 8226and U266 cell-lines was achieved with each of the antibodies (FIG. 2A).These findings were further validated by showing the ability ofanti-MUC1 SP mAb SPmAb-2.1 and the MUC1 TRA mAb H23 to specificallystain ES-2 MUC1 negative ovarian cells following transfection with theMUC I-TM construct (FIG. 2B). The MUC1-TM construct contains the entireMUC1 i.e. the alfa plus beta subunits and hence it includes the TRA andthe SP domains in case that it is not chopped by signal peptidase in theER In summary, these results confirmed MUC1-associated membrane bindingto each of the MUC1's SP antibodies as observed in the flow cytometryexperiments.

TABLE 3 Human Cell- Mouse SPmAb- Rabbit lines Origin control HH23SPmAb-6 2.1 control R23 ES-2 Ovarian Carcinoma 234 32 248 237 251 247OVCAR-3 Ovarian Carcinoma 370 456 11440 1019 418 10800 MCF7 BreastCarcinoma 230 004 1091 413 236 4705 MDA-453 Breast Carcinoma 182 80 559290 165 2443 MDA-231 Breast Carcinoma 245 66 591 312 215 1931 RajiB-Lymphoblastic 131 03 307 179 128 519 Leukemia Ramos B-Lymphocytic 198660 402 111 384 Leukemia U266 Multiple Myeloma 485 039 661 574 471 1716RPMI8226 Multiple Myeloma 745 585 1190 1154 738 2331 ARH-77 Plasma cell176 68 691 439 161 3966 Leukemia SK-mel- Melanoma 117 19 114 112 113 11728 SK-mel-1 Melanoma- 352 25 342 345 350 348 Metastasis

The human B-Lymphocytic leukemia lines Raji and Ramos, the human MM celllines U266, and RPMI 8226 and the human Plasma cell Leukemia line ARH-77were grown in suspension in RPMI-1640 medium supplemented with 10% FBS,L-Glutamine, Sodium Pymvate, non-essential amino-acids, HEPES andGentamycin (Biological Industries, Israel). The human ovarian carcinomaline OVARCAR-3 was grown as adherent monolayer culture in RPMI-1640medium supplemented with 15% FBS, L-Glutamine, Sodium Pymvate andGentamycine. The human ovarian carcinoma line ES-2 and melanoma linesSK-mel-28 were grown as adherent monolayer cultures and the humanMelanoma line SK-mel-1 was grown as suspension culture in DMEM medium,supplemented with 10% FBS L-Glutamine, Sodium Pymvate and Gentamycine.All cell lines were purchased from the American Type Culture Collection(ATCC, Manassas, USA). As a positive control for MUCl expression, theanti-MUCl TRA mAb H23 was used. This monoclonal antibody was raisedagainst the human breast cancer cell line T47D 16 and recognizes theepitope APDTRP on the non-glycosylated form of MUCl. As a negativecontrol in FACS analysis, mice anti goat or rabbit ant-mouse IgG-FITCconjugated were used (Jackson immunoResearch, USA).

Flow cytometry for cell lines was performed as follow: 1×10⁷ evaluatedcells were washed once with PBS and incubated for 30 min in stainingbuffer consisting of PBS supplemented with 3% FBS, 10% Hu AB sera (i.e.pooled human sera obtained from donors) and 0.1% sodium acid in FACStubes, over-night.

Next, buffer was removed by centrifugation and 50 μg/sample of FITCconjugated Abs were added to different tubes for 30 min at R.T. Labeledcells were washed twice, fixed in BD CellFIX (Becton Dickenson, USA)according to the manufacturer's protocol and stored at 4° C. untilanalyzed. Four-color flow cytometry analysis was performed on the LSR II(Becton Dickenson Immunocytometry Systems, USA) and the data wereanalyzed using FlowJo software (TreeStar, USA).

Conjugation of antibodies with FITC was performed as followed: 50 μ1 ofFITC (1 mg/ml solution. Sigma, IL) diluted in DMSO (Sigma, IL) wereadded to 0.5 ml of IgG (2 mg/ml) diluted in 0.1M sodium bicarbonatebuffer at pH=9 and incubated, with stirring, for 8 hat 4° C. At the endof this incubation step, the FITC-IgG conjugation was dialyzed againstPBS for 48 h at 4° C.

Example 4 Expression of MUCl SP and MUCl on Solid Tumors as Assayed byImmunohistochemistry

The ability of anti-MUC1-SP antibodies to diagnose solid tumors wasevaluated by immunohistochemistry analysis of breast cancer tissue inparaffin blocks (prepared by the Pathology Department at HaddasahUniversity Hospital, Jerusalem). The Results, presented in FIGS. 3A-3Bare an example of the positive staining observed in sections taken froman infiltrating duct carcinoma of the breast and demonstrate specificstaining of tumor tissue obtained from breast cancer patient with H23(FIG. 3A) and SPmAb-2.1 (FIG. 3B). As can be seen, the number of stainedcells per tumor section varied as well as the intensity of staining percell, thereby confirming specificity.

Immunohistochemistry analysis was performed on de-paraffinized tissueslices using H23 or SPmAb-2.1 MUC I antibodies at a final concentrationof 50 μg/ml for 1 h at room temperature. PolyScan HRP/DAB detectionsystem kit (Cell Marque, USA) was then used according to the recommendedprotocol.

Example 5 Expression of MUCl SP and MUCl on BM Cells of MM Patients

Evaluating the expression levels of SP epitopes present on tumor cellsobtained from a patient is a valuable tool in selecting subjects thatmay benefit from treatment with a vaccine comprising MUCI SP, such asImMucin [Kovjazin, R. et al. Vaccine (2011)]. The vaccine comprises anSP domain of a protein and was shown to raise a powerful specific immuneresponse against said SP domain.

In the following example it is demonstrated that the antibodies of theinvention are capable of binding to cell surface expressed MUC1 SP onbone marrow cells of patients suffering from multiple myeloma (MM).

The ability of R23IgG polyclonal antibodies to selectively bind tumorcells was investigated in an ex-vivo setting with malignant plasma cellsfound in bone marrow aspirates obtained from three patients withmultiple myeloma (MM). The heterogeneous cell population in theaspirates allows direct assessment of MUC1-specific SP (using the R23IgGantibody) and MUC1-TRA (using the H23 antibody). These antibodies areexpected to bind MM cells that express MUC1, while minimally binding tonon-MUC1 expressing cells that are present in the same aspirate samples.

The binding properties of R23IgG and H23 antibodies, in terms ofintensity (per sample) and frequency (%) were compared among differentMM patients, using FACS analysis. Bone marrow aspirates (2-3 ml) weredrawn from 3 patients (50-75 years) with slowly progressing asymptomaticMM.

FACS analysis of BM cells was performed as follows: I ml of BM cellsobtained from a patient was incubated in 15 ml polypropylene tubes(CellStar, Greiner, Fricknhausen, Germany), for 15 min at RT, in thepresence of 2 mM EDTA Then, IO ml (IO-fold diluted in Double DistilledWater) of BD FACS lysing Solution (Catalog #349202, IOX) was added tothe cells, for further incubation of 10 min (RT). Next, the remainingcells were washed with staining buffer, using a centrifugation step of 5min at 1500 RPM, and placed in FACS tubes (BD Falcon™, Franklin LakesN.J. USA). Cells were then blocked for 15 min with staining buffer,supplemented with 10% AB serum (Sigma Israel, Rehovot, Israel). Stainingwas performed using APC-conjugated antibodies CD138 (IQP, Groningen,Netherlands), eFluor450-Kappa, PE-Lambda (ebiosciences, San-Diego,Calif., USA) and FITC-conjugated (Sigma Israel, Rehovot, Israel) H23,R23IgG, which were added for an incubation step of 30 min, at RT, in thedark. Following this step, cells were washed with 2 ml of stainingbuffer and re-suspended in 0.5 ml PBS. Samples were then analyzed usinga LSR II FACS (BD Biosciences, San Jose, Calif., USA).

A population of “large cells” was initially gated by side vs. forwardscatter (FIG. 4A) their phenotype as plasma cells was verified bystaining with anti-Kappa light chain-eFluor 450 and anti-Lambda-PElabeled antibodies, as detailed below. This gated cells population wasnext analyzed for MUC1 TRA (FIG. 4C) and MUC1 SP (FIG. 4E) expression onCD138 positive MM cells. Anti-CD138-APC labeled antibody was used forthis analysis, to further select MM cells, and the R23IgG and H23antibodies were labeled with FITC fluorophores. In each experimentspecies matched control antibodies were used for MUC1 staining, eithernormal mouse IgG-FITC conjugated (FIG. 4B), or normal rabbit IgG-FITCconjugated (FIG. 4D).

The results with freshly obtained bone marrow aspirates from 2 MMpatients (P#I and P#II) revealed R23IgG immunoreactivity in 78.2% and66.3% of the two CD138 positive cell populations (FIG. 4E, column E) andH23 immunoreactivity in 78.3% and 59.2% of the two CD138 positive cellpopulations (FIG. 4C, column C). The specificity of the staining washigh, with marginal staining for both CD138 positive/MDC I negativecells and to species matched control antibodies.

In contrast, the expression levels of MUC1 TRA and SP (based on theresults obtained with H23 and R23IgG, respectively) were low in thethird aspirate (P#3), 0.37%, and 0.45% respectively, while CD138expression levels in this aspirate was still moderate (74.9% and 39.9%).In these three patients, the R23IgG and H23 antibodies seem to recognizeMUC1 SP and MUC1 TRA (respectively) in the same population of malignantplasma cell although the staining intensity with R23IgG vs. H23 was upto one log higher. The analysis in this study was part of a screeningprocess performed for enrolment into a phase 1/11 clinical trial of thecancer vaccine ImMucin (protocol VAXIL-001). The study was approved bythe Ethics Committee of Haddasah University hospital, Jerusalem, Israeland the Israeli Ministry of health.

Importantly, the results obtained with R23IgG provide a tool forselecting patients which may benefit from treatment with an agentdirected against the MUC1 SP epitope, in particular, with a therapeuticvaccine comprising the MUC1 SP (e.g. ImMucin).

In addition, another plausible therapeutic approach would be to targetMUC1 SP positive cancer cells using anti-MUC1 SP antibodies optionallyconjugated to anti cancer agents.

Example 6 Expression of MUC1 SP and MUC1 on BM of MM Patients as Assayedby Immunohistochemistry

The specificity of R23IgG as compared with H23 antibodies was furtherconfirmed using Immunohistochemistry, by staining fresh samples preparedfrom BM aspirates of MM patients. As shown in FIGS. 5A and 5B, specificbinding of both antibodies to plasma cells was observed (the positivestained cells are indicated by an arrow). However, while H23 (FIG. 5A)manifested strong binding, which was mainly localized inside the cells,staining with R23IgG (FIG. 5B) was more delicate and was mainlylocalized on the cell membranes. The results obtained in theimmunochemistry analysis were consistent with the results obtained inthe FACS analysis of the BM cells, which were obtained for the samepatients and thus reconfirmed their specificity.

Immunohistochemistry analysis was performed as follows: BM samplesderived from patients were placed on slides, air-dried and fixed for 10min at −20° C., with acetone. Staining with H23 and R23IgG antibodieswas performed according to the protocol of the HRP/DAB Detection SystemCat# CMQ 95 ID (Cell Marque, USA) with additional staining with Giemsasolution (MERCK HX888942) at the final step.

In summary, binding was demonstrated for both types of antibodies thosedirected against the MUC1 SP and those directed to the non-SP epitopeTRA, in solid and in non-solid MUC1 positive tumor cells, reconfirmingthe ability of anti MUC I SP antibodies to detect MUC1 presentation onthe cell surface of cells. Furthermore, binding of R23IgG was found tobe stronger and more specific than the binding of H23 (which is directedto a non-SP portion of MUC1), particularly in the case of MM/B-celllines.

Example 7 Evaluation of Anti-MUC1 SP Antibodies in a ComplementDependent Cytotoxicity (CDC) Assay

In the following example, the functional ability of the antibodies ofthe invention to recognize and affect tumor cells bearing the antigenwas tested by means of tumor specific lysis.

Complement dependent cytotoxicity (CDC) analysis using the rabbitpolyclonal antibodies R23IgG (FIG. 6A) and the two monoclonalantibodies, namely, SPmAb-2.1 and SPmAb-6 (FIG. 6B) demonstrated astrong lysis of MUC1 expressing cells. As demonstrated in FIG. 6A,R23IgG polyclonal antibodies manifested 60-100% lysis of solid tumorsOVACAR-3 ovarian cells, and of the non-solid tumors, U266, RPMI 8226,MM, ARH-77 and Ramos Leukemia tumor cells.

In a similar manner, as demonstrated in FIG. 6B, SPmAb-2.1 and SPmAb-6manifested strong specific lysis of >90% and 60-70%, respectively of thesame cell lines. In these experiments H23 demonstrated a similar trendof 80-90% lysis compared to SPmAb-2.1 and SPmAb-6 (FIG. 6B). The lysisby each of the anti-MUC1 antibodies was highly statistically significant(p>0.001 or more, t-test), with respect to the MUC1 expressing celllines vs. MUC1-negative ovarian cell line ES-2 and melanoma cell lineSK-mel-1. Moreover, the negative control, such as normal mouse sera(NMS), Normal mouse IgG (NM IgG) and normal rabbit sera (NRS) allmanifested significantly lower percentage of lysis (see FIGS. 6A and6B). Generally, the CDC lysis efficacy positively correlates with theMUC1 cell surface expression levels evaluated by flow cytometry analysis(Table 3). One exception from this observation was found with mAbSPmAb-6, which demonstrated high cell surface binding and lower CDC. Thepotential explanation for this difference can be related to itsIg-gamma2a isotype which was previously shown to be less active thanlg-gamma I in CDC [Watier, H. et al., Transplantation 62(1): 105-113(1996) and Chuntharapai, A et al., J. Immunol., 166(8):4891-4898(2001)].

CDC analysis was performed as described above, with the followingchanges: Cells were incubated with

the antibodies H23, R23IgG, SPmAb-6 or SPmAb-2.1 (100, 50 and 10 μg/ml).Plates (96-well) were from Griner (De Groot, Germany) and a PerkinElmerBeta-counter was used for evaluation (IL USA). In addition, normal miceor rabbit IgG antibodies were used (Chemicon, Millipore, USA). Theresults were statistically analyzed with student's t-test. In all tests,the minimum level of significance for a 2-tailed test was set at P<0.01.

Example 8 Sera Expression Levels of sMUC1 in Healthy Donors and CancerPatients

Expression levels of soluble MUCI (sMUC1, MUCI Ag, or sMUC1 TRA) in serasamples, obtained from healthy donors and cancer patients, weredetermined using the commercial M4H2 anti-TRA monoclonal antibody, whichrecognizes the core antigen of soluble MUC1.

Levels of sMUC1 were evaluated using ELISA. Briefly, the ELISA protocolincluded ELISA plates (F96 Maxisorp, Nunc, Roskild, Denmark), acommercial anti-MUC1 monoclonal antibody that was raised against a TRApeptide and recognizes soluble MUCl's core antigen (clone M4H2), and anELISA kit (HyTest, Turku, Finland), which was used according to themanufacturer's protocol. sMUC1 levels were evaluated using seven serialI 00μ1 dilutions of patient's sera, starting at a 1:5 dilution. As aMUC1 positive control, six dilutions (starting at a I:5 dilution) ofsupernatant collected from the DA-3™ cell line were used. The ELISAplates were developed with TMB/E solution (CHEMICON, Millipore,Billerica, Mass., USA). The reactions were terminated by the addition of50μ1/well of 10% sulfuric acid. The results were measured at 450 nm. Incases where a pure antigen or specific antibody was not used asstandards, this assay consisted of measuring the “Specific titer” ratherthan the absolute concentration.

The Tables below summarize the results obtained for naïve healthy donors(Table 4), cancer patients with solid tumors (Table 5), each of whichhaving a different disease stage and a different tumor indication(mainly Colon and Rectal cancers), and multiple myeloma patients (Table6) with various stages of MM.

TABLE 4 sMUC1 and anti-MUC1 autoantibodies levels in naïve donors sMUC1Anti-MUC1 Anti-MUC1 Sample Sample TRA sMUC1 SP TRAAb SP Ab No.characterization (μg/ml) ¹ (μg/ml) ¹ (μg/ml)2 (μg/ml)2 1 Naïve healthydonor Negative Negative 224 178 2 Naïve healthy donor Negative Negative265 159 3 Naïve healthy donor Negative Negative 184 145 4 Naïve healthydonor Negative Negative 161 185 5 Naïve healthy donor Negative Negative205 185 6 Naïve healthy donor Negative Negative 230 200 7 Naïve healthydonor Negative Negative 281 216 8 Naïve healthy donor Negative Negative318 10 9 Naïve healthy donor Negative Negative 105 110 10 Naïve healthydonor Negative Negative 320 166 11 Naïve healthy donor Negative Negative203 10 12 Naïve healthy donor Negative Negative 147 200 13 Naïve healthydonor Negative Negative 200 184 14 Naïve healthy donor Negative Negative134 100 15 Naïve healthy donor Negative Negative 152 10 Average 208.60137.20 Standard deviation 65.35 72.97 ¹ Sera levels of MUC1 antigen andanti-MUC1 antibodies were measured by ELISA assay as described above.Positive sMUC1 level is set for titer of X > 1:5. 2Naïve sera foranti-MUC1 TRA antibodies epitope is X . . . : S, 274 μg/ml and foranti-MUC1 SP autoantibodies is X . . . : S, 210.2 μg/ml, based on theaverage plus standard deviation values determined in 15 naïve healthyindividuals.

TABLE 5 Anti- Anti- Anti- VXL25 VXLl00 VXL3A Anti- MUCl Ag Ab Ab AbVXL211 Patient Indication (titer) (μg/ml) (μg/ml) (μg/ml) Ab (titer) B#1Colorectal 0 294 1481 333 >1:100 B#2 Colon 1:20 382 1781 708 >1:100 B#3Colorectal 1:20 266 2016 520 >1:100 B#4 Colorectal 1:5  260 3744625 >1:100 B#6 Lung 1:5  816 3136 1041 >1:100 B#7 Colon 0 47 113238 >1:100 B#9 Chorionic 1:40 267 465 362 >1:100 B#10 Prostate 0 290 460375 >1:100 B#12 Colon 375.33 1523.67 516.89 Average 249.46 1279.43245.74 STDEV

TABLE 6 sMUC1 and anti-MUC1 autoantibodies levels in multiple myelomapatients Anti- Anti- Anti- MUC1 Anti- sMUC1 sMUC1 MUCl MUC1 TRAAb MUClSP Patient TRA SP TRAAb SP Ab (% Ab (% No. Patient status (titer)1(titer) (μg/ml)² (μg/ml)² Pos.) Pos.) 1 Active disease; under NegativeNegative 440 740 + + therapy 2 Progressive disease; Negative Negative183 150 under therapy 3 At best response; off 1:10 Negative 382 312 + +therapy 4 Active disease; under 1:5 Negative 190 350 + therapy 5 Activedisease; under 1:10 Negative 85 50 therapy 6 Active disease; underNegative Negative 97 130 therapy 7 At best response; off 1:10 Negative355 456 + + therapy 8 At best response; off Negative Negative 211 581 +therapy 9 Active disease; under Negative Negative 132 1036 + therapy 10At best response; off Negative Negative 158 746 + therapy 11 Progressivedisease; Negative Negative 238 525 + under therapy 12 Progressivedisease; Negative Negative 63 500 + under therapy 13 At best response;off Negative Negative 500 1000 + + therapy 14 Active disease; under 1:10Negative 292 884 + + therapy 15 Progressive disease; 1:40 Negative 9803400 + + under therapy 16 At best response; off Negative Negative 7611500 + + therapy 17 Progressive disease; 1:20 Negative 91 100 undertherapy 18 Progressive disease; Negative Negative 728 424 + + undertherapy 19 Progressive disease; 1:10 Negative 795 339 + + under therapy20 Progressive disease; Negative Negative 120 80 under therapy 21Progressive disease; 1:20 Negative 3200 812 + + under therapy 22Progressive disease; Negative Negative 291 594 + + under therapy 23Progressive disease; Negative Negative 500 732 + + under therapy 24Progressive disease; 1:10 Negative 500 191 + under therapy 25Progressive disease; 1:20 Negative 860 950 + + under therapy 26 Activedisease; under 1:20 Negative 195 162 therapy 27 Progressive disease; 1:5Negative 222 976 + under therapy Percentage of patients with positiveanti-MUC1 SP and MUC1 TRA specific 14/27 20/27 antibodies levels 1Seralevels of MUC1 antigen and anti-MUC1 antibodies were measured by ELISAassay as described above. Positive sMUC1 level is set for titer of X >1:5 ²Naïve sera for anti-MUC1 TRA antibodies epitope of X ≤ 274 μg/mland for anti MUC1 SP autoantibodies is X ≤ 210.2 μg/ml, based on theaverage plus standard deviation values determined in 15 naive healthyindividualsAs demonstrated in Table 4, expression of sMUC1 in sera samples obtainedfrom naïve healthy donors was low to undetectable, referred to as“negative” (at a titer level of less than 1:5). However, the level ofsMUC1 was significantly higher (up to 8-fold, at a titer level of ≤1:40)in a large portion of patients. These results are consistent with thecurrent knowledge of soluble MUC1 expression in naïve healthy donors andin cancer patients, having MUC1 positive tumors.

Example 9 Sera Expression Levels of MUCl SP in Healthy Donors and CancerPatients

The expression levels of soluble MUC1 SP in sera samples obtained fromnaïve healthy donors and from cancer patients were determined using theSPimAb-3A anti-SP polyclonal antibody, in an ELISA assay, as describedabove. SPimAb-3A anti-SP polyclonal antibody is an IgG fraction ofpolyclonal hyper immune sera (obtained from rabbit), which is directedagainst the MUC1 SP-derived peptide, VXLI00, which consists of thecomplete SP of MUC-1.

The results obtained for naïve healthy donors and for MM patients, whichare at various disease stages are summarized in Tables 4 and 6.Expression of soluble MUC1 SP, as measured using the R23 antibody, wasnot observed neither in the sera of naïve healthy donors or in the seraof MM patients. Namely, there is no correlation between disease stageand the level of soluble SP fragments of MUC1 or the level of theendogenously generated antibodies produced against cellular/surface SP.

Example 10 Sera Expression Levels of Endogenous Anti-MUCl Antibodies inHealthy Donors and Cancer Patients

The expression levels of endogenously generated antibodies to peptidesderived from MUC1 were analyzed. Particularly, expression levels ofendogenous antibodies which recognize VXL25 (a peptide derived from MUC1TRA) as well as endogenous antibodies which recognize VXLI00 and VXL3A(peptides derived from MUC1 SP), were evaluated in sera samples obtainedfrom naïve healthy donors and cancer patients.

The expression levels of the endogenously generated antibodies describedabove 30 were evaluated using an ELISA assay. Briefly, ELISA plates (F96Maxisorp, Nunc, Roskild, Denmark) were activated by 0.1% ofglutaraldehyde in carbonate buffer pH 9 for 1 h at RT and coated with50μ1 of MUCl-TRA-L peptide at 5 μg/ml in carbonate buffer and incubatedovernight at 4° C. Plates were then blocked with 200 μl of PBSsupplemented with 0.5% gelatin for 2 hat 25° C. Evaluated sera sampleswere then diluted 1:100 in PBS with 0.5% gelatin and incubated for 2 hat 25° C. Next, 50 μl/well of the appropriate secondary anti-IgGantibody HRP-conjugate (CHEMICON, Millipore, Billerica, Mass., USA) wasadded at a final dilution 1:10,000 in a blocking buffer and incubatedfor 1 h at 25° C. Plates were then developed as described above. For apositive standard, we used 6 double dilutions starting from 10 μg/ml ofthe anti-TRA mAb H23. In this assay, naïve sera for MUCl are X<200μg/ml, based on a mean value determined from 10 healthy individuals.

The monoclonal antibody (mAb) H23 was used as a positive control, bypreparing six double dilutions (starting from 10 μg/ml) of thisantibody. The monoclonal antibody H23 was raised against the humanbreast cancer cell line T47D, and recognizes the APDTRP epitope of thenon-glycosylated form of MUC1 TRA (Keydar I, et al. Proc Natl Acad SciUSA 1989, 86: 1362-1366). H23 is capable of recognizing soluble MUC1 TRAdomain in sera or on cancer cells. It also recognizes the 25-mer peptideVXL25 (derived from MUCl TRA, as described before).

The results obtained for naïve healthy donors and cancer patients withdifferent cancers and at different stages of the disease are presentedin Tables 4-6.

As demonstrated in Table 4, expression levels of 137.2±72.97 and208.6±65.35 μg/ml were observed for anti-MUCl SP endogenous antibody(which binds to VXL3A), and anti-MUCl TRA antibodies, respectively, innaïve healthy donors. Surprisingly, the expression levels of antibodiesto the MUCl peptides, namely anti-VXL3A, and anti-VXL25 (TRA), weresignificantly elevated (3-7 folds) for the different cancer patients(Tables 5 and 6), while the expression level of antibodies to thenon-TAA, Tuberculosis-derived SP VXL211 was very low (Table 5). Theseresults confirm the existence of endogenously generated antibodies toMUC1 SP in cancer patients.

Importantly, a high concentration of anti-MUC1 SP autoantibodies wasalso present in patients with minimal disease (characterized as‘patients at best response off therapy’), four of which (namely,patients number 8, 10, 13 and 16) had undetectable sMUCl levels,suggesting a potential role for these anti-MUCl SP autoantibodies indetecting a disease at an early stage and possibly the disease onset.

The preferred immunogenicity of the MUC1 SP vs. the MUC1 TRA domain canbe further demonstrated using the same group of patients, in which theconcentration of the anti-SP autoantibodies was significantly higher(P<0.03, t-test) than that of the anti-MUC1 TRA levels. For thesepatients, any potential influence on sMUCl levels by anti-s MUC1 TRAautoantibodies was ruled out with a dedicated ELISA that analyzed theamount of sMUC1 in antigen-antibody complexes. This further supports theimmunodominant properties of MUCl SP regarding antibody production.

The percentage of patients having a “positive” anti-MUC1 SP or MUC1TRA-specific titers was further analyzed. A positive response wasdefined for the average titer plus one standard deviation, as describedbelow.

As a positive standard for anti-MUC1 TRA antibodies, dilutions startingwith 10 μg/ml of the anti-MUC1 TRA mAb H23 [Keydar, I. et al. PNAS86:1362-1366 (1989)] were used. These antibodies were raised against thehuman breast cancer cell line T47D and recognized the TRA epitope APDTRPon the non-glycosylated form of MUC1. As a positive standard foranti-MUCI SP antibodies, dilutions beginning with I0 μg/ml ofanti-MUC1-SP-M rabbit polyclonal antibodies were used. In this assay,serum levels for anti-MUCl TRA autoantibodies in naïve donors wereX:S274 μg/ml and for anti-MUC1 SP autoantibodies, X:S210.2 μg/ml, basedon the average plus standard deviation value determined in 15 naïvehealthy individuals.

The resulting analysis presented in Table 6 further supports the initialobservation of an elevated production of anti-MUC1 SP autoantibodies. Inparticular, most patients, 20/27 (74%) had positive anti-MUC1SP-specific autoantibodies, while only 14/27 (51.8%) had positiveanti-MUC1 TRA-specific autoantibodies. These differences demonstrated apositive trend for the selectivity of MUCl's SP vs. TRA domain inmultiple myeloma patients.

1 Since, as mentioned above, expression of MUCl SP was not observed inthe sera of naïve healthy donors or MM patients, the target antigen (orepitope) for the endogenously generated anti-MUC1 SP could possibilitybe expressed on the cell surface of cancer cells. Expression of MUCl SPon cell surface may be either as an independent molecule and/or inassociation with MHC molecules.

The low expression levels of antibodies obtained for the non-TAA SPVXL211 (derived from the MTb protein, Rv0476/MTO4941) in naïve donorsand in cancer patients, is consistent with the expression of MUCl SP asa cancer-specific marker (or target) rather than an SP non-specificresponse.

Interestingly, the most significant results, having the lowest standarddeviations, were detected in the case of the MUC1 SP peptide VXL3A, inall types of cancers tested. Consistently, the difference in theexpression levels of endogenously generated anti-VXL3A antibodiesbetween naïve healthy donors and cancer patients of all cancer types hasthe highest statistical difference, as verified by at-test shown inTable 7 (0.0007-0.01). A similar analysis, performed for anti-VXL100endogenous antibodies showed that while the difference in the expressionlevels obtained for naïve healthy donors and for cancer patients wasless prominent than in the case of the anti-VXL3A endogenous antibodies,the difference was still significant, mainly in sera samples of patientswith solid tumors (Table 7).

TABLE 7 T-Test Anti VXL25 AntiVX100 Anti VXL3A (two tails) Ab Ab Ab s0.05426591 0.02541475 0.0101179 B 0.04110985 0.00850219 0.0007251 H0.05924422 0.04359874 0.0030535

Significantly, these results suggest that the VXL3A sequence, whichconsists of the 10-21 C-terminal 12 amino acids of the 21 amino acidspeptide VXL100, is of greater specificity for the endogenously generatedantibodies. The lower variability obtained for the endogenouslygenerated antibodies to the VXL3A peptide is consistent with thesefindings.

Inferior results were obtained with the same sera samples for the MUCTRA peptide. According to the statistical t-test conducted in this case,the difference in the concentration of endogenously generated anti-VXL25antibodies is of (marginal) significance only for patients with solidtumors (Table 7, 0.008). These results are consistent with previouspublications, which showed a high variation in the levels ofendogenously generated antibodies to MUC1 TRA sequences, such as VXL25,in cancer patients.

Example 11 Correlation Between Sera Expression Levels of EndogenousAnti-MUCl SP Antibodies and sMUCl

The levels of sMUCl (MUCl Ag) were compared with the levels ofendogenously generated antibodies against VXL3A, VXL100 and VXL25, asdescribed above, for a group of 25 cancer patients having non-solidtumors (MM, FIGS. 7A-7C) and for 10 cancer patients having solid tumors,in particular Colon, Rectal, Lung, and Prostate (FIGS. 7D-7F). Asdemonstrated in FIG. 7A a positive correlation was observed for the seralevels of sMUCl and the sera level of endogenously-generated anti-VXL3Aantibodies, for MM patients (R²=0.475 for all 25 patients assayed). Mostinterestingly, a higher correlation (R²=0.7606) was demonstrated forpatients having an advanced stage of the cancer disease (16 out of the25 patients).

Lower correlations, of R²=0.165 and R²=0.081, were found forendogenously-generated anti-VXL25 antibodies (FIG. 7B) and anti-VXL100antibodies (FIG. 7C), respectively, within the same assay and patients.

As demonstrated in FIGS. 7D-7F, a similar analysis performed withpatients with solid tumors manifested no correlation between the levelsof sMUC1 and the levels of endogenously generated antibodies against anyof the peptides, namely VXL25, VXLl00, VXL3A (R²=0.011, 0.0091 and 0.004respectively).

These results can possibly be related to the large diversity in thelevels of sMUC1 in patients with solid tumor vs. patients with non-solidtumor. Particularly, these results may possibly be attributed to thefact that most of the patients analyzed in this group had surgery fortumor removal, which may potentially result in the reduction of thelevel of sMUCl.

Example 12 Correlation Between Sera Levels of the Different Anti-MUClAntibodies

The half-life of endogenously generated antibodies in the sera ofpatients is relatively prolonged and is not immediately influenced bythe levels of the antigen. The concentrations of the various anti-MUClpeptide antibodies were inter-correlated, as described above. Theresults obtained for patients with solid tumors (FIGS. 8C and 8D) showeda positive correlation between the titer levels of anti-VXL3A andanti-VXL25 antibodies (D) (R²=0.45 1 1). However, no correlation(R²=0.0733) was found between the titer levels of anti-VXL25 andanti-VXl00 antibodies (C).

Similar analysis conducted for MM patients (FIGS. 8A and 8B) manifestedan inverse correlation pattern. While a positive correlation (R²=0.5665)was observed for the titer levels of anti-VXL25 and anti-VX:l 00antibodies (FIG. 8A), no correlation was found between the titer levelsof anti-VXL3A and anti-VXL25 antibodies (FIG. 8B) (R²=0.0992). Theseresults may potentially be used for developing a tool for earlydetection of MUC1 positive tumors, before disease onset, or fordeveloping a tool for monitoring disease progression.

Example 13 Sera Expression Levels of Endogenous Antibodies to MUCl SP inBRCA Carriers

A BRCA mutation is a mutation in either of the genes BRCAI or BRCA2.Harmful mutations in these genes produce hereditary breast-ovariancancers in affected families. Hundreds of different types of mutationsin these genes have been identified. Women with harmful mutations ineither BRCAI or BRCA2 have risk of breast cancer that is about fivetimes the normal risk, and a risk of ovarian cancer that is about ten tothirty times the normal risk.

However, at present, the available diagnostic tests do not enable thedetection of all of these mutations. In addition, early detection ofbreast and ovarian cancers is limited, since appropriate markers thatsignal mutations in the BRCA genes are currently not available.

The expression levels of endogenously generated antibodies whichrecognize VXL25 (a peptide derived from MUC1 TRA) as well as endogenousantibodies which recognize VXLI00 and VXL3A (peptides derived from MUC1SP), were evaluated in sera samples obtained from BRCAI/2(−) andBRCAI(+) and BRCA2(+) carriers.

As demonstrated in FIG. 9B, the expression levels of endogenouslygenerated antibodies which recognize VXLI00 were significantly higher inBRCAI (+) and BRCA2 (+) carriers (n=29, 21, respectively) than inBRCAI/2(−) carriers (n=15), with the t-test values p=0.001 and p<0.001,respectively. Similarly, as demonstrated in FIG. 9C, the expressionlevels of endogenously generated antibodies which recognize VXL3A weresignificantly higher in BRCAI (+) and BRCA2 (+) carriers than inBRCAI/2(−)

30 carriers (p<0.001). No significant difference was found between thelevels of endogenously generated antibodies which recognize VXL25 inBRCAI (+), BRCA2 (+) or BRCAl/2(−) carriers (FIG. 9A). The mean age forall the subjects tested was 38 (20-73 years).

The correlation between the levels of endogenous antibodies directed toMUC1 SP and the presence of the BRCAI and/or BRCA2 gene mutation showthat the autoantibody levels of these antibodies may be used fordetection of cancers associated with BRCA mutations, e.g. breast andovarian cancers.

Example 14 Levels of Endogenous Antibodies Directed to Non-MUC1 TAA SPin Sera of Cancer Patients

The endogenously generated antibody titers to SP domains of various TAAswere evaluated as described above, in particular for ARMET (VXLI0I),Uroplakin II (VXL104), PAP (VXL106) and Mammaglobin-1 (VXL108). Theanalysis was performed for MM patients (Table 8 A) and patients withsolid tumors (Table 8 B). Results demonstrated that endogenouslygenerated antibodies exist also for these TAA SPs, and these antibodiesmay thus be used for diagnosis as discussed above.

TABLE 8 Sera levels of antibodies to non-MUC1 TAA SP in cancer patientsAnti Anti Anti Anti Anti VXLl00 VXL101 VXL104 VXL106 VXL108 PatientIndication (titer) (titer) (titer) (titer) (titer) A H#2 MM 1:3200 1:2001:100 1:100 1:100 H#3 MM 1:3200 1:200 1:100 1:100 1:100 H#S MM 1:128001:200 1:100 1:100 1:100 H#8 MM 1:1600 1:200 1:100 1:100 1:100 B 8#2Colon 1:3200 1:200 1:200 1:200 1:100 8#3 Colorectal 1:6400 1:200 1:2001:200 1:100 8#4 Colorectal 1:6400 1:200 1:200 1:200 1:100 8#7 Colon1:800 1:200 1:200 1:400 1:100 8#9 Chorionic 1:200 1:800 1:400 1:8001:400

Example 16 Levels of Antibodies to SP in Sera of Tuberculosis Patients

The expression levels of endogenously generated antibodies to fiveimmunogenic SP domains from key antigens in MTb were analyzed,particularly, Antigen 85B (VXL201), Lipoprotein lpqH (VXL203), ATPdependent helicase putative (VXL 208), Uncharacterized proteinRv0476/MTO4941 precursor (VXL 211) and Uncharacterized proteinRv1334/MT1376 precursor (VXL 212).

SP selection for these assays was based on the immunodominant propertiesof these MTb peptides, particularly, the strong proliferation ability ofthe SP domains on a large pool of naïve healthy donors and MTb patients,and further, the high IL2 secretion by stimulated T cells, obtained fromnaïve donors, that correlated with theirs helper function to supportantibody production (Kovjazin, R. et al., Mol Immunol 2011,48:1009-1018).

Table 9 presents the results obtained for seven naïve healthy donors andfor seven patients having active tuberculosis. The results are presentfor individual samples and also as an average±standard deviation for thenaïve healthy donors as well as for the patients. In naïve healthydonors (Table 9, B) expression levels of 285±146, 257.6±97, 214±134,185±146 and 185±146 were obtained, for anti-VXL201, VXL203, VXL208,VXL211 and VXL211 endogenous antibodies, respectively. The concentrationof endogenously generated antibodies to all five MTb SP Vaccinecandidates was found to be elevated (3.2-39.5 folds) in tuberculosis(MTb) patients (Table 9, A).

TABLE 9 sera levels of antibodies to SP in tuberculosis patients andNaïve donors VXL201 VXL203 VXL208 VXL211 VXL212 A P#l 1600 3200 128001600 800 P#3 800 3200 12800 800 800 P#l3 1600 3200 12800 1600 800 P#14400 800 1600 400 400 P#17 400 1600 3200 800 400 P#18 800 1600 12800 1600800 P#19 800 1600 3200 800 400 Average 914.29 2171.43 8457.14 1085.71628.57 STDVE 501.43 1002.85 5442.69 501.43 213.81 B ND#l 100 200 100 100100 ND#2 200 200 200 100 100 ND#3 100 200 200 100 100 ND#4 400 200 100100 100 ND#S 400 200 100 100 100 ND#6 400 400 400 400 400 ND#7 400 400400 400 400 Average 285.71 257.14 214.29 185.71 185.71 STDVE 146.3997.59 134.52 146.39 146.39 Ratio P/N 3.20 8.44 39.47 5.85 3.38

These results confirm the existence of endogenously generated antibodiesto the five SP domains (particularly directed to the peptides VXL208 andVXL203) in tuberculosis patients.

Since in the case of VXLl00 and VXL3A, low expression levels of SPfragments in the sera of naïve healthy donors were observed, it isplausible that the target antigen/epitopes for the endogenouslygenerated SP may be expressed on the cell surface of bacteria and/or onMTb infected cells. Thus, SP expression may be either observed as anindependent molecule and/or in association with MHC molecules.

Example 17 Induction of Humoral Response to SP of Various MTb Proteinsin Mice

In a set of in vivo experiments, the immunogenicity and synergisticproperties of a number of combinations of the five MTb SP Vaccinecandidates (VC) referred to above in Example 16 (i.e. VXL201, VXL203,VXL208, VXL 211 and VXL212) were evaluated. Thus, 7 weeks old BALB/cmice were vaccinated two or three times at weekly intervals with a totalof 100 μg per mouse of the following mixtures:

Mixture 1: VXL201, VXL203, VXL208, VXL211 and VXL212; Mixture 2: VXL201,VXL203, VXL21 l and VXL212; Mixture 3: VXL201 and VXL 203; and Mixture4: VXL21 land VXL 212. The mixtures were dissolve in DMSO (Sigma AldrichIsrael/PBS and administered to mice without the addition of a carrier oran adjuvant. Even in the absence of an adjuvant, a hyper immune sera wasunpredictably generated to few of the SP mixtures, mainly to Mixture 3(VXL201 and VXL203), as demonstrated in Table 10. In addition, thesesurprising results were obtained after merely two vaccinations.

TABLE 10 Induction of humoral response to MTb SP in BALB/c mice A Mix1 IMix1 I Mix2 I Mix2*2 Mix3 I Mix I Mix4 I Evaluated *3 *2 *3 Mix3*3 II *24*3 *2 PBS Peptide Serum titer VXL201 1:800 1:400 1:1600 1:1600 1:32001:3200 1:100 1:100 1:100 VXL203 1:800 1:400 1:1600 1:1600 1:3200 1:32001:100 1:100 1:100 VXL208 1:400 1:400 1:800 1:400 1:800 1:400 1:100 1:1001:100 VXL211 1:400 1:400 1:400 1:400 1:100 1:100 1:100 1:100 1:100VXL212 1:400 1:400 1:400 1:400 1:100 1:100 1:100 1:100 1:100 B MouseVXL201 VXL203 VXL211 1 1:24000 1:1600 1:200 2 1:24000 1:3200 1:200 31:1600 1:3200 1:200 4 1:24000 1:800 1:200

This humoral response (Table 10) did not interfere with a robust T-cellresponse to these mixtures, as evaluated by proliferation andcytotoxicity. The results shown in Table 10A demonstrate a significantelevation in the anti-VXL201 and in the anti-VXL203 titers, following 2or 3 vaccinations with Mixture 3. Based on these results, thevaccination regimen was repeated (3 times) at weekly intervals in fourBALB/c mice, using only Mixture 3. The results obtained in thisexperiment (Table 10B) showed a highly significant and specific titer,of up to 1:24,000 in three out of the four mice tested, mainly toVaccine candidates (VC) VXL201. The titer to VXL203, the second peptidein this Mixture, was specific, but significantly lower than thatobtained for VXL201. On the other hand, the response to VXL211 VCevaluated in this experiment as a control SP did not induce any antibodytiter.

Example 18 Antibodies Recognize VXL201 and VXL203 on the Cell Surface ofMTb Cells

As shown above, vaccination of mice with Mixture 3 was able to generatespecific antibodies that recognize VXL201 and to some extent alsoVXL203. The following example demonstrates that these epitopes arespecifically expressed on MTb bacterial cells and are recognized by theantibodies on the cell surface.

Hyperimmune sera with high titer were used for immunofluorescencestaining of MTb bacteria (FIG. 10D), as well as for staining of arelated mycobacterium strain, the M. Kansasii (FIG. 10E). The existenceof bacteria in the preparations was confirmed using4′-6-Diamidino-2-phenylindole (DAPI) DNA staining (FIGS. 10A-10C). Theresults clearly showed specific binding by the hyper immune sera only inthe case of the MTb bacteria (upper Right), while no staining of theMycobacterium Kansasi was observed (middle right panel). In addition, nobinding was observed for MTb, when sera from normal mice were used (FIG.10F).

1. A method for detecting cell surface expression of a MUC1 signalpeptide (SP), the method comprising: a. obtaining a biological samplecontaining cells from a subject; and b. determining the expression levelof said MUC1 SP, by contacting a surface of cells in said biologicalsample with an antibody directed against a first 11 amino acids of saidMUC1 SP, or any fragment thereof, and detecting the binding of saidantibody and said first 11 amino acids of said MUC1 SP.
 2. The method ofclaim 1, wherein said antibodies are directed against a peptide selectedfrom a group consisting of the peptides denoted by: SEQ ID NO: 3, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:
 10. 3.The method of claim 2, wherein said antibody is directed against apeptide denoted by SEQ ID NO:
 3. 4. A method for treatment of a subjectsuffering from a disease, the method comprising administering to saidsubject a therapeutically effective amount of at least one antibodydirected against a first 11 amino acids of a MUC1 SP, or any fragmentthereof.
 5. The method of claim 4, wherein said antibodies are directedagainst a peptide selected from a group consisting of the peptidesdenoted by: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 9, and SEQ ID NO:
 10. 6. The method of claim 5, wherein saidantibody is directed against a peptide denoted by SEQ ID NO:
 3. 7. Themethod of claim 4, wherein said disease is cancer, a bacterial disease,a fungal disease, a parasitic disease, a prion disease, or a viraldisease.
 8. The method of claim 7, wherein said disease is cancer. 9.The method of claim 8, wherein said cancer is MUC1 positive cancer. 10.The method of claim 4, wherein said antibody is an antibody thatactivates the complement system, or activates cellular cytotoxicity. 11.The method of claim 4, wherein said antibody is associated with orcombined with a cytotoxic moiety.
 12. The method of claim 11, whereinsaid cytotoxic moiety is selected from a radioactive agent, a toxin, ananti-metabolite, a chemotherapeutic agent, a drug, a growth inhibitoryagent and an alkylating agent.
 13. The method of claim 4, wherein saidantibody is associated with or combined with an imaging agent.
 14. Themethod of claim 13, wherein said imaging agent is a fluorophore.
 15. Akit, comprising: (a) at least one isolated antibody directed against afirst 11 amino acids of a MUC1 signal peptide (SP), or any fragmentthereof; (b) means for detecting the binding of said isolated antibodyto said MUC1 signal peptide; and (c) optionally instructions for use ofsaid kit.
 16. The kit of claim 15, wherein said antibody is directedagainst a peptide selected from a group consisting of the peptidesdenoted by: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 9, and SEQ ID NO:
 10. 17. The kit of claim 16, wherein saidantibody is directed against a peptide denoted by SEQ ID NO:
 3. 18. Thekit according to claim 15, wherein the kit further comprises a vaccinecomprising the signal peptide, or a fragment thereof capable ofeliciting an immune response, to said MUC1 SP.
 19. The kit of claim 15,for diagnosing cancer.
 20. The kit of claim 19, wherein said cancer isMUC1 positive cancer.